Vaccine delivery method

ABSTRACT

A vaccine delivery method is presented that includes a composition including as one component a slurry matrix that is a liquid at room temperature and a gel at physiological pH, physiological salt concentrations and/or physiological temperatures and as a second component one or more antigens. Also included are methods of inducing an immune response in a subject and vaccinating a subject by administering such compositions.

CONTINUING APPLICATION DATA

This application is a divisional application of U.S. patent applicationSer. No. 17/061,696, filed Oct. 2, 2020, which is a continuationapplication of U.S. patent application Ser. No. 15/411,083, filed Jan.20, 2017, which is a continuation application of U.S. patent applicationSer. No. 13/793,329, filed Mar. 11, 2013, which is acontinuation-in-part of International Application No. PCT/US2012/034012,filed Apr. 18, 2012, which claims the benefit of U.S. ProvisionalApplication Ser. No. 61/476,431, filed Apr. 18, 2011, all of which areincorporated by reference herein in their entireties.

GOVERNMENT FUNDING

This invention was made with government support under AI071883 andAI036657 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submittedvia EFS-Web to the United States Patent and Trademark Office as an ASCIItext filed entitled “23501840121_SequenceListing_ST25.txt” having a sizeof 17 kilobytes and created on Dec. 29, 2016. The information containedin the Sequence Listing is incorporated by reference herein.

BACKGROUND

Vaccines remain the single greatest public health asset for combatinginfectious diseases. The goal of vaccine delivery is to present vaccineantigens in a manner that enhances antigen presenting cell activation,uptake of antigen and processing. An additional goal is to reduce thenumber of vaccinations required to induce an effective, vaccine-specificresponse, especially if a single effective dose of a vaccine isavailable. Current, conventional vaccine delivery methods use alum.Aluminum salts, such as alum, were first licensed for use as adjuvantsin human vaccines in the 1920's. There is a need for improved deliverymodes and adjuvants that are safe for use in vaccine formulations.

SUMMARY OF THE INVENTION

The present invention includes a composition including as one componenta slurry matrix that is a liquid at room temperature and a gel atphysiological salt concentrations and/or physiological temperatures andas another component one or more antigens. In some aspects of thecomposition, the slurry matrix is a peptide hydrogel. In some aspects,the peptide hydrogel includes PURAMATRIX, or a derivative thereof. Insome aspects, the peptide hydrogel includes the peptide scaffoldRADARADARADARADA (SEQ ID NO:2), or a derivative thereof. In some aspectsof the composition, the slurry matrix includes MATRIGEL, or a derivativethereof.

In some aspects of the composition, the composition further includes oneof more adjuvants. In some aspects, an adjuvant includes a Toll-LikeReceptor (TLR) agonist and/or a cytokine. In some aspects, a TLR agonistincludes a TLR4 agonist. In some aspects, a TLR agonist includes a TLR9agonist. In some aspects, a TLR9 agonist includes a CpGoligodeoxynucleotide (ODN).

In some aspects of the composition, the composition further includes aToll-Like Receptor (TLR) agonist and/or a cytokine. In some aspects, aTLR agonist includes a TLR4 agonist. In some aspects, a TLR agonistincludes a TLR9 agonist. In some aspects, a TLR9 agonist includes a CpGoligodeoxynucleotide (ODN).

In some aspects of the composition, the antigen includes a hepatitisantigen, an influenza antigen, a schistosomiasis antigen, and/or aburkholderia antigen, or an antigenic fragment thereof.

The present invention includes a method of producing an immune responsein a subject, the method including administering a composition asdescribed herein to the subject.

The present invention includes a method of immunizing a subject, themethod including administering a composition as described herein to thesubject.

The present invention includes a method of delivering one or moreimmunogenic antigens to a subject, the method including administering acomposition as described herein to the subject.

The present invention includes a method of delivering one or moretherapeutic antigens to a subject, the method including administering acomposition as described herein to the subject.

In some aspects of the methods of the present invention, the subject isa domestic livestock or a companion animal. In some aspects of themethods of the present invention, the subject is poultry. In someaspects of the methods of the present invention, the subject is human.

In some aspects of the methods of the present invention, administrationof the composition includes subcutaneous (sc) injection, intramuscular(im), intradermal, mucosal, intraperitoneal (ip), and aerosol delivery.

In some aspects of the methods of the present invention, administrationof the composition includes administration as a primary and/or boostervaccination.

In some aspects of the methods of the present invention, administrationof the composition includes administration as a booster vaccinationafter a primary vaccination with a polypeptide vaccine or a plasmid DNAvaccine.

The present invention includes a method of producing an anti-schistosomeimmune response in a bovoid, the method including administering acomposition including as one component a slurry matrix that is a liquidat room temperature and is a gel at physiological conditions and asanother component one or more schistosome antigens to the bovoid.

The present invention includes a method of producing an anti-schistosomeimmune response in a bovoid, the method including administering acomposition as described herein to the bovoid, wherein one or moreantigen includes a schistosome antigen.

The present invention includes a method of schistosomiasis vaccinationin a bovoid, the method including administering a composition includingas one component a slurry matrix that is a liquid at room temperatureand is a gel at physiological conditions and as another component one ormore schistosome antigens.

The present invention includes a method of schistosomiasis vaccinationin a bovoid, the method including administering a composition asdescribed herein to the bovoid, wherein one or more antigen includes aschistosome antigen.

In some aspects of the methods, the composition further includes one ormore adjuvants. In some aspects, an adjuvant includes a Toll-LikeReceptor (TLR) agonist and/or a cytokine. In some aspects, a TLR agonistincludes a TLR4 and/or a TLR9 agonist. In some aspects, a TLR9 agonistincludes a CpG oligodeoxynucleotide (ODN). In some aspects, a CpG ODNincludes bovine CpG. In some aspects, the adjuvant includes the cytokineIL-12.

In some aspects of the methods, the composition further includes aToll-Like Receptor (TLR) agonist and/or a cytokine. In some aspects, aTLR agonist includes a TLR4 and/or a TLR9 agonist. In some aspects, aTLR9 agonist includes a CpG oligodeoxynucleotide (ODN). In some aspectsof the methods, a CpG ODN includes bovine CpG.

In some aspects of the methods, the schistosome antigen includes aSchistosoma japonicum antigen, or an antigenic fragment thereof.

In some aspects of the methods, the schistosome antigen includes is aSjCTPI polypeptide, a SjCTPI-Hsp70 polypeptide, a SjC23 polypeptide,and/or a SjC23-Hsp70 polypeptide, or an antigenic fragment thereof.

In some aspects of the methods, administration of the compositionincludes administration as a primary and/or a booster vaccination.

In some aspects of the methods, administration of the compositionincludes administration as booster vaccination after a primaryvaccination with a SjCTPI-Hsp70 plasmid DNA vaccine.

In some aspects of the methods, the method further includesadministration of one or more anti-schistosome chemotherapeutic agents.

In some aspects of the methods, the method demonstrates at least 45%efficacy in the prevention of infection with a schistosome parasite.

The present invention includes methods of making the compositiondescribed herein.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the kinetics of IgA anti-HBsAg antibody titers. Data shownis pooled from two independent experiments for total n=10.

FIG. 2 shows the kinetics of IgM anti-HBsAg antibody titers. Data shownis pooled from two independent experiments for total n=10.

FIG. 3 shows the kinetics of IgG anti-HBsAg antibody titers. Data shownis pooled from two independent experiments for total n=10.

FIG. 4 shows the kinetics of IgG₁ anti-HBsAg antibody titers. Data shownis pooled from two independent experiments for total n=10.

FIG. 5 shows the kinetics of IgG_(2a) anti-HBsAg antibody titers. Datashown is pooled from two independent experiments for total n=10.

FIG. 6 shows higher anti-HBsAg antibody titers after single vaccination(21 days). Data shown is pooled from two independent experiments fortotal n=10; *p<0.05, **p<0.01, **p<0.001, ****p<0.0001 compared toantigen alone using two-way ANOVA with Bonferroni post-test.

FIG. 7 shows higher anti-HBsAg antibody titers after single vaccination(35 days). Data shown is pooled from two independent experiments fortotal n=10; *p<0.05 compared to antigen alone using two-way ANOVA withBonferroni post-test.

FIG. 8 shows cytokine profile twenty-four hours after HBsAgre-stimulation of splenocytes. Data shown is representative of oneexperiment, n=5; *p<0.05, ****p<0.0001 compared to antigen alone usingtwo-way ANOVA with Bonferroni post-test.

FIG. 9 shows cytokine profile forty-eight hours after HBsAgre-stimulation of splenocytes. Data shown is pooled from two independentexperiments for total n=10 (n=5 for adjuvants only); no statisticaldifferences seen (p<0.05) compared to antigen alone using two-way ANOVAwith Bonferroni post-test.

FIG. 10 shows cytokine profile seventy-two hours after HBsAgre-stimulation of splenocytes. Data shown is pooled from two independentexperiments for total n=10 (n=5 for adjuvants only); **p<0.01 comparedto antigen alone using two-way ANOVA with Bonferroni post-test.

FIG. 11 shows increased HBsAg-specific cell-mediated immunity by ELISpotin HbsAg-specific T cell responses. Data shown is pooled from twoindependent experiments for total n=10; *p<0.05 compared to antigenalone using two-way ANOVA with Bonferroni post-test.

FIG. 12 shows increased HBsAg-specific T cell-mediated immunity by flowcytometry. Data shown is pooled from two independent experiments fortotal n=10; **p<0.01 compared to antigen alone using two-way ANOVA withBonferroni post-test.

FIG. 13 is a radar graph representing the prevalence of ex vivo cytokinebalance in a range of cell subsets from immunized mice (n=10/group) 24hours post-primary inoculation. The values of the axis can be joined toform the central polygon area which represents the general cytokinebalance. The analysis of the radar chart axes highlights thecontribution of leukocyte for the overall cytokine balance.

FIGS. 14A and 14B are radar graphs representing the prevalence of exvivo cytokine balance in a range of cell subsets from immunized mice(n=10/group) 48 hours post-primary inoculation. FIG. 14A presents TNFand IFN gamma. FIG. 14B presents IL-5 and IlL-4. Each axis displays theproportion of each cytokine balance category. The values of each axiscan be joined to form the central polygon area which represents thegeneral cytokine balance. Increasing or decreasing central polygon areasreflect higher or lower contribution of inflammatory or regulatorycytokine balance in each group.

FIG. 15 is a radar graph representing the prevalence of ex vivo cytokinebalance in a range of cell subsets from immunized mice (n=10/group) 72hours post-primary inoculation. The values of the axis can be joined toform the central polygon area which represents the general cytokinebalance. The analysis of the radar chart axes highlights thecontribution of leukocyte for the overall cytokine balance.

FIGS. 16A to 16D are comparisons of rHepBag-specific antibodies inducedin immunized mice (n=10/group) between 14 and 35 days post-primeinoculation. The boost was performed 28 days after prime immunizationand rHepBag-specific antibodies levels were determined by ELISA assay.FIG. 16A presents data from 14 days, FIG. 16B presents data from 21days, FIG. 16C presents data from 28 days, and FIG. 16D presents datafrom 35 days. Statistical significance at P<0.05 are represented bysuperscript letters ‘a’, ‘b’ and ‘c’ for comparisons with rHepBag,rHepBag in ALHYDROGEL® and rHepBag in Freund, respectively.

FIGS. 17A to 17D show IgG1:IgG2a ratio after immunization of mice(n=10/group) with rHepBag plus adjuvants between 14 and 35 days. Theboost was performed 28 days after prime immunization andrHepBag-specific antibodies levels were determined by ELISA assay. FIG.17A presents data from 14 days, FIG. 17B presents data from 21 days,FIG. 17C presents data from 28 days, and FIG. 17D presents data from 35days.

FIGS. 18A and 18B show increased anti-NP IgG titers in two strains ofmice vaccinated with rNP. FIG. 18A shows titers in C57BL/6 mice. FIG.18B shows titers in Balb/c mice. Sera were collected four weekspost-last vaccination (4wplv).

FIGS. 19A and 19B show increased anti-influenza IgG titers in in C57Bl/6mice vaccinated with PR8 WIV. FIG. 19A and FIG. 19B represent resultsfrom two independent experiments. Sera were collected four weekspost-last vaccination (4wplv).

FIGS. 20A and 20B show increased protection from lethal challenge inC57Bl/6 mice vaccinated with PR8 WIV. FIG. 20A shows results from anindependent experiment with lethal challenge at 30 LD₅₀. FIG. 20B showsresults from an independent experiment with lethal challenge at 1000LD₅₀.

FIGS. 21A to 21C show increased anti-burkholderia IgG titers in miceimmunized with a cocktail of three recombinant burkholderia proteinantigens with three different adjuvants (alum, CFA, or PURAMATRIX® gel).FIG. 21A shows anti-burkholderia protein 4-9 IgG titers in immunizedmice. FIG. 21B shows anti-burkholderia protein 22-11 IgG titers inimmunized mice. FIG. 21C shows anti-burkholderia protein 42 IgG titersin immunized mice.

FIGS. 22A and 22B show IgG anti-schistosome CCA protein antibody titersin mice immunized with CCA in complete Freund's adjuvant (FIG. 22A) andCCA in MATRIGEL plus CpG (FIG. 22B).

FIGS. 23A to 23C show increased anti-NP influenza antibody titers usingstandard vaccination methodologies. FIG. 23A shows a schematic of astandard method including a prime vaccination and a boost vaccination.Baseline corrected anti-NP endpoint titers at 6 weeks post vaccinationare shown for both C57BL/6 (FIG. 23B) and BALB/C (FIG. 23C) strains ofmice; *p<0.05, **p<0.01 using a 1-way ANOVA with Tukey's multiplecomparison test.

FIGS. 24A to 24C show greatly increased anti-PR8 influenza antibodytiters after single vaccination. FIG. 24A shows a schematic of a newvaccination delivery system including only a single vaccination. FIG.24B and FIG. 24C show anti-PR8 endpoint titers at 4 weeks postvaccination; **p<0.01, ***p<0.001, ****p<0.0001 using a 1-way ANOVA withBartlett's test for equal variances and Bonferroni's multiple comparisontest.

FIGS. 25A and 25B show anti-influenza IgG titers. FIG. 25A showsbaseline corrected, influenza-specific IgG, sera titers at 4 weekspost-last vaccination. FIG. 25B baseline corrected, influenza-specificIgG endpoint titers at 4 weeks post-last vaccination. **p<0.01,***p<0.001, ****p<0.0001 using a 1-way ANOVA with Bartlett's test forequal variances and Bonferroni's multiple comparison test.

FIGS. 26A to 26D show an anti-PR8 influenza-specific antibody classanalysis of pre-challenge sera at 4 weeks post vaccination. FIG. 26Ashows anti-influenza IgE baseline corrected endpoint titers. FIG. 26Bshows anti-influenza IgA baseline corrected endpoint titers. FIG. 26Cshows anti-influenza IgM baseline corrected endpoint titers. FIG. 26Dshows anti-influenza IgG baseline corrected endpoint titers. *p<0.05,**p<0.01, compared to new method (PR8+X+CpG) using nonparametric 1-wayANOVA with Kruskal-Wallis and Dunns multiple comparison post-test.

FIGS. 27A to 27D show an anti-PR8 influenza-specific antibody IgGsubtype analysis of pre-challenge sera at 4 weeks post vaccination. FIG.27A shows anti-influenza IgG-λ-1 baseline corrected endpoint titers.FIG. 27B shows anti-influenza IgG-2a baseline corrected endpoint titers.FIG. 27C shows anti-influenza IgG-2b baseline corrected endpoint titers.FIG. 27D shows anti-influenza IgG-3 baseline corrected endpoint titers.*p<0.05, **p<0.01, ***p<0.001, compared to new method (PR8+X+CpG) usingnonparametric 1-way ANOVA with Kruskal-Wallis and Dunns multiplecomparison post-test.

FIGS. 28A to 28C show decreased morbidity following lethal, homologousflu challenge. FIG. 28A shows percent body weight after lethal challengeat 1,000 LD₅₀. FIG. 28B shows body score after lethal challenge at 1,000LD₅₀. FIG. 28C shows percent survival after lethal challenge at 1,000LD₅₀; *p<0.05, ***p<0.001, compared to naive using Log rank (Mantel-Cox)with Gehan-Breslow-Wilcoxon test.

FIGS. 29A and 29B show viral clearance following homologous lethalchallenge. FIG. 29A shows a schematic of a method including vaccinationwith whole inactivated virus, lethal challenge at 4 weeks postvaccination, and clearance assessments at 1, 2, 3, and 5 days postchallenge. FIG. 29B shows enhanced kinetics of viral clearance of PR8from the lungs at 1, 2, 3, and 5 days post challenge followinghomologous lethal challenge at 1,000 LD₅₀; **p<0.01, ****p<0.0001compared to naïve at the same time point using two-way ANOVA.

FIGS. 30A and 30B show mucosal IgG antibody titers in lung homogenatesfollowing challenge. FIG. 30A shows baseline corrected,influenza-specific IgG sera titers in lung homogenates at 1 daypost-challenge. FIG. 30B shows influenza-specific IgG whole lungendpoint titers in lung homogenates at 1 day post-challenge.

FIGS. 31A and 31B show mucosal IgA antibody titers in lung homogenatesfollowing challenge. FIG. 31A shows baseline corrected,influenza-specific IgA sera titers in lung homogenates at 1 daypost-challenge. FIG. 31B shows influenza-specific IgA whole lungendpoint titers in lung homogenates at 1 day post-challenge.

FIGS. 32A and 32B show inter-strain reactivity of anti-HA IgG antibodiesin sera. FIG. 32A shows baseline corrected, anti-HA IgG HI-(A/Ca/07/09)sera titers in lung homogenates at 4 weeks post-vaccination with PR8WIV. FIG. 32B shows endpoint anti-HA IgG HI-(A/Ca/07/09) sera titers inlung homogenates at 4 weeks post-vaccination with PR8 WIV.

FIGS. 33A and 33B show inter-strain reactivity of anti-HA IgG antibodiesin sera. FIG. 33A shows baseline corrected, broadly reactive Anti-HA IgGH5-(A/HK/156/97) sera titers in lung homogenates at 4 weekspost-vaccination with PR8 WIV. FIG. 33B shows endpoint anti-HA IgGH5-(A/HK/156/97) sera titers in lung homogenates at 4 weekspost-vaccination with PR8 WIV.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS OF THE INVENTION

The present invention provides compositions and methods that provide forimproved vaccine delivery, providing for the improved focal andsustained delivery of antigen at the site of vaccine administration;enhancing antigen presenting cell activation and antigen uptake andprocessing. With the present invention, one or more immunogenic agentsis administered in a composition including a slurry matrix componentthat is a liquid at room temperature, non-physiological pH, and/or lowsalt concentrations and a gel at physiological salt concentrationsphysiological pH, and/or physiological temperatures. Gelling may beinduced by the physiological body temperature of a vertebrate, such as amammal or bird. Such a temperature may be, for example, at least about25° Celsius (C), at least about 30° Celsius, at least about 32° Celsius,at least about 35° Celsius, at least about 37° Celsius, at least about39° Celsius, or at least about 40°. Gelling may be induced by thephysiological salt concentrations. In some embodiments, gelling may inthe presence of millimolar concentrations of salt, for example by a saltconcentration of greater than about 0.05 molar (M). Gelling may beinduced by the physiological pH of a vertebrate, such as a mammal orbird.

Thus, the vaccine composition gels or polymerizes after administrationto a subject, localizing the vaccine antigens to a single site whereinnate antigen presenting cells can home and begin taking up vaccineantigens. Ideally, the slurry matrix is a biocompatible material willnot induce undesirable reactions in the body as a result of contact withbodily fluids or tissue, such as tissue death, tumor formation, allergicreaction, foreign body reaction (rejection), inflammatory reaction,antibody response, or blood clotting, for example. A slurry matrix mayalso be referred to herein as a “biomedical polymer hydrogel,” a“biomedical hydrogel,” “biomedical polymer,” a “polymer hydrogel,” a“biocompatible polymer hydrogel,” a “biocompatible hydrogel,” a“biocompatible polymer,” or a “hydrogel.” As used herein, a “hydrogel”is a 3-dimensional network of cross-linked, hydrophilic macromoleculescapable of being swelled and incorporating about 20 percent to about 95percent water by weight. A hydrogel is a gel in which the liquidconstituent is water. As used herein, a gel is a solid, jelly-likematerial that can have properties ranging from soft and weak to hard andtough. A gel is a substantially dilute cross-linked system, whichexhibits no flow when in the steady-state. By weight, gels are mostlyliquid, yet they behave like solids due to a three-dimensionalcross-linked network within the liquid. It is the crosslinks within thefluid that give a gel its structure (hardness). In this way gels are adispersion of molecules of a liquid within a solid in which the solid isthe continuous phase and the liquid is the discontinuous phase. Hydrogelis a network of polymer chains that are hydrophilic, sometimes found asa colloidal gel in which water is the dispersion medium. Hydrogels arehighly absorbent (they can contain over 99.9% water) natural orsynthetic polymers. Hydrogels also possess a degree of flexibility verysimilar to natural tissue, due to their significant water content.

Any of a wide variety of biomedical polymer hydrogels available for usein medical technologies may be used with the methods and compositionsdescribed herein.

In some embodiments, the slurry matrix is naturally occurring, such asfor example, fibrin, collagen, elastin, agarose, methylcellulose,hyaluronan, and other naturally derived polymers. In some embodiments,the slurry matrix is MATRIGEL, or a derivative thereof. MATRIGEL is abasal membrane extract from mouse cells and includes laminin, collagenIV, entactin, nidogen, and proteoglycans. The invasion of tumor cellsinto MATRIGEL has been used to study the involvement of extracellularmatrix receptors and matrix degrading enzymes in tumor progression andinvasion and MATRIGEL has been used also as an in vitro and in vivoangiogenesis model (MATRIGEL plug assay) to study the activity ofangiogenic and anti-angiogenic cytokines and other substances. MATRIGELis commercially available as BD MATRIGEL™ Matrix. See the world wide webat bdbiosciences.com/cellculture/ecm/ecmtypes/index.jsp.

In some embodiments, a slurry matrix is synthetic, such as for example,a synthetic peptide hydrogel or a self-assembling peptide (sapeptide)scaffold. The sapeptide scaffolds are formed through the spontaneousassembly of ionic self-complementary beta-sheet oligopeptides underphysiological conditions, producing a hydrogel material. These shortpeptides (typically about 8, about 12, about 16, about 24, or about 32amino acid residues with internally-repeating sequences) self-assemblein aqueous salt solution into three-dimensional matrices. The peptidesare characterized as being amphiphilic, having alternating hydrophobicand hydrophilic amino acid residues; greater than 12 amino acids, andpreferably at least 16 amino acids; complementary and structurallycompatible. Complementary refers to the ability of the peptides tointeract through ionized pairs and/or hydrogen bonds which form betweentheir hydrophilic side-chains, and structurally compatible refers to theability of complementary peptides to maintain a constant distancebetween their peptide backbones. Peptides having these propertiesparticipate in intermolecular interactions which result in the formationand stabilization of beta-sheets at the secondary structure level andinterwoven filaments at the tertiary structure level. Examples include,but are not limited to, peptide family members, RAD16-I ((RADA)(4); SEQID NO:2), RAD16-II ((RARADADA)(2); SEQ ID NO:3), KFE-8 ((FKFE)(2); SEQID NO:4), or KLD-12 ((KLDL)(3); SEQ ID NO:5). See, for example, U.S.Pat. No. 5,670,483; Holmes et al., 2000, Proc Natl Acad Sci USA;97(12):6728-33; Yokoi et al., 2005, Proc Natl Acad Sci USA;102(24):8414-9; Liu et al., 2012, Nanoscale; 4(8):2720-7, and BDPURAMATRIX™ Peptide Hydrogel, Guidelines for Use, Catalog Number 354250(SPC-354250-G rev 2.0; BD Biosciences, Bedford, Mass.). In some aspects,a peptide hydrogel includes the peptide scaffold self-assemblingbuilding blocks of arginine-alanine-aspartate-alanine (RADA; SEQ IDNO:1). In some aspects, a peptide hydrogel includes RADARADARADARADA(SEQ ID NO:2), or a derivative thereof. In some aspects, the peptidehydrogel includes PURAMATRIX, or a derivative thereof. See, for example,U.S. Pat. No. 5,670,483; Holmes et al., 2000, Proc Natl Acad Sci USA;97(12):6728-33; Yokoi et al., 2005, Proc Natl Acad Sci USA;102(24):8414-9; Liu et al., 2012, Nanoscale; 4(8):2720-7, and BDPURAMATRIX™ Peptide Hydrogel, Guidelines for Use, Catalog Number 354250(SPC-354250-G rev 2.0; BD Biosciences, Bedford, Mass.), each of whichare incorporated herein in their entireties.

In some embodiments, a biomedical polymer hydrogel may be a polyethyleneglycol (PEG) hydrogel that polymerizes spontaneously in vivo.

In some embodiments, a slurry matrix, in addition to gelling atvertebrate or mammalian body temperature and/or physiological saltconcentrations, is a bioresorbable synthetic polymer that degrades anddissolves with time. Such compounds are naturally degraded in the bodyby hydrolysis and absorbed as water-soluble monomers. Examples include,polylactic acid, polylactide (PLA), poly (L-lactic acid),poly-D-lactide, polyglycolic acid (PGA), polyglycolide and itscopolymers (poly(lactic-co-glycolic acid) with lactic acid, homo- andcopolymers of lactic acid and glycolic acid, poly (DL-lacticacid/glycine) copolymers, poly (DL-lacticco-glycolic acid) (PLGA), poly(DL-lacticco-glycolic acid) (PLGA), porous poly(DL-lactic-co-glycolicacid) foams, poly(amino acids) poly[ox(1-oxo-1,2-ethanediyl)]((C₂H₂O₂)_(n); Biovek), poly(glycolide-co-caprolactone),poly(glycolide-co-trimethylene carbonate), polydioxanone (PDO, PDS),poly-p-dioxanone, caprolactone (also referred to as 2-oxepanone),epsilon-caprolactone, 6-hexanolactone, hexano-6-lactone,1-oxa-2-oxocycloheptane polyglactin 910, polyanhydrides, andpolyorthoester films formed from poly (D,L-lactic-co-glycolic acid,88:12) (PLGA) or from a 50/50 (w/w) blend of PLGA and poly (L-lacticacid) (PLLA). See, for example, Schakenraad and Dijkstra, 1991, ClinMater; 7(3):253-69; Mooney et al., 1997, J Biomed Mater Res;37(3):413-20; and Lu et al., 2000, Biomaterials; 21(18):1837-45.

Compositions of the present invention include one or more antigenicagents (also referred to herein as an immunogen). An antigenic agent maybe any of the great variety of agents that are administered to a subjectto elicit an immune response in the subject. An antigenic agent may bean immunogen derived from a pathogen. The antigenic agent may be, forexample, a peptide or protein antigen, a viral antigen or polypeptide,an inactivated virus, a recombinant virus, a bacterial or parasiticantigen, an inactivated bacteria or parasite, a whole cell, agenetically modified cell, a tumor associated antigen or tumor cell, atoxin, a lipid, a glycolipid, a glycoprotein, or a carbohydrate antigen.In some applications an antigen is not a living cell. In someembodiments, an antigenic agent is a soluble antigen.

A composition as described herein may include as an antigenic agent anyof the great variety of immunogenic agents available as vaccinecomponents. Such vaccines may include, but are not limited to, antigenicvaccines components directed against various infectious, viral, andparasitic diseases, toxins, and anti-tumor vaccine components. Antitumorvaccines include, but are not limited to, peptide vaccines, whole cellvaccines, genetically modified whole cell vaccines, lipid vaccines,glycolipid vaccines, glycoprotein vaccines, recombinant protein vaccinesor vaccines based on expression of tumor associated antigens byrecombinant viral vectors. In some embodiments, an antigenic agent is asoluble antigen.

As shown in Examples 8-11, the use of a composition as described hereinin vaccination methods to deliver a vaccine antigen unexpectedly andsurprisingly resulted in improved results, demonstrating significantlyincreased induction of specific antibodies, enhanced antibody titers,improved antibody isotype profiles, induction of a mixed Th1-Th2response, increased protection from challenge, decreased morbidityfollowing challenge, decreased mortality following challenge, anddramatically enhanced pathogen clearance.

An antigenic agent includes a bacterial or viral antigen from, forexample, diptheria, Streptococcus pneumoniae, Staphylococcus aureus,Bacillis anthracis (such as, for example, PA), Haemophilus influenzae,Kliebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa,Moraxella catarrhalis, Coxiella burnetii, Mycoplasma pneumoniae,Legionella pneumophila, Chlamydophila pneumoniae, Yersinia pestis andYersinia enterocolitica, Hantavirus and other Bunyaviruses, Rhodococcus,Corynebacteria, adenovirus, parainfluenza, respiratory syncitial virus,coronavirus (SARS-CoV), varicella zoster virus, Herpes zoster,cytomegalovirus, cholera, enterotoxigenic Escherichia coli,enterohemorrhagic Escherichia coli, Helicobacter pylori, rotavirus,Salmonella spp Listeria, Human immunodeficiency virus, Herpes virus,mumps, measles (paramyxovirus, Morbillivirus, Rubella), chicken pox,polio, sexually transmitted diseases (Chlamydia, gonorrhea, genitalherpes, human papilloma virus, syphilis, bacterial vaginosis,Trichomoniasis, candidiasis, Treponema pallidum, tuberculosis(Mycobacteria spp.), rocky mountain spotted fever, Yellow fever, Dengueand other Flaviruses, Filoviruses such as Ebola and Marburg, babesia,viral hepatitis (Hepatitis A, B, C, E), Clostridium botulinum,Francisella tularensis, Burkholderia pseudomallei and mallei, Brucellaspecies, Typhus Fever (Rickettsia prowazecki), Shigella species,Cryptosporidium parvum, Norwalk virus, Pathogenic Vibrios, Arenaviruses,Campylobacter jejuni, Caliciviruses, Microsporidia, Cyclospora spp.,West Nile Virus, Lacrosse virus, California encephalitis virus,Venezuelan equine encephalitis, Eastern Equine encephalitis, WesternEquine encephalitis, Japanese Encephalitis virus, Nipah Virus, Prions,Chikungunya virus, tickborne encephalitis viruses, tickborne hemorrhagicfevers viruses, influenza virus types A or B, seasonal and pandemicinfluenza vaccines.

An antigenic agent may be a toxin, such as, for example, ricin toxinfrom Ricinus communis, epsilon toxin from Clostridium perfringens,Staphylococcus enterotoxin B, aflatoxin from Aspergillus flavus, snakevenoms, insect venoms, fish venoms, and plant toxins.

An antigenic agent may be an antigenic agent of a sexually transmitteddisease (STD), such as for example, human immunodeficiency virus (HIV),herpes, and human papillomavirus (HPV).

An antigenic agent includes a parasite antigen from, for example, amalaria parasite or a schistosome parasite. Malaria antigens include,but are not limited to antigens from the plasmodium species Plasmodiumvivax, Plasmodium falciparum, and Plasmodium knowlesi, Plasmodium ovale,and Plasmodium malariae. Schistosome parasites include, but are notlimited to, Schistosoma japonicum, Schistosoma mansoni, and Schistosomahaematobium. A schistosome antigen may be a schistosome triose phosphateisomerase (CTPI) protein, or antigenic fragment or derivative thereof,including, but not limited to a S. japonicum, S. monsoni, or S.haematobium CTPI protein, or antigenic fragment or derivative thereof. Aschistosome antigen may be a schistosome tetraspin 23 kDa integralmembrane protein (C23), or antigenic fragment or derivative thereof,including, but not limited to a S. japonicum, S. monsoni, or S.haematobium C23 protein, or antigenic fragment or derivative thereof.Such a schistosome antigen may be a chimeric polypeptide, fused to oneor more additional antigenic determinants, such as for example, a heatshock protein, or antigenic fragment or derivative thereof, including,but not limited to, bovine heat shock protein 70 (Hsp70). Schistosomeantigens include, for example, SjCTPI, SjCTPI-Hsp70, SjC23, andSjC23-Hsp70 polypeptides. Additional schistosome antigens include, forexample, paramysosin, glutathione S-transferase, omega-1, fatty acidbinding protein, molecules involved with binding to or transport ofinsulin, and sugars such as glucose, fatty acids, cholesterol, CAA, CCA.

Antigens may be from any of a variety of other parasites, including, butnot limited to, kinetoplastid protozoa, such as for example, protozoa ofthe Blastocrithidia, Crithidia, Endotrypanum, Herpetomonas, Leishmania,Leptomonas, Phytomonas, Trypanosoma, and Wallaceina genera. In preferredembodiments, the protozoan is of the genus Trypanosoma, including, butnot limited to, T cruzi, T brucei, Tb. gambiense, and Tb. rhodesiense.In some embodiments, the protozoan is of the genus Leishmania,including, for example, Leishmania major. Notable trypanosomal diseasesinclude trypanosomiasis (African Sleeping Sickness and South AmericanChagas Disease, caused by species of Trypanosoma) and leishmaniasis(caused by species of Leishmania). In some embodiments, a viral antigenis, for example, toxoplasma, giardia, Entamoeba spp., schistosomiasis,onchocerchiasis, other filarial nematodes, gastroinstestinal nematodessuch as ascaris, strongyloides, cestodes such as Taenia spp., andEchinococcus spp.

An antigenic agent includes a viral antigen from, for example,hepatitis, such as for example, hepatitis C, hepatitis B, or hepatitisA, influenza, for example, the M2, hemaglutinin, and/or neuraminidaseproteins of an influenza virus, including, for example, influenza A(including, but not limited to, the H5N1 and H1N1 subtypes), influenzaB, and influenza C, respiratory syncytial virus (RSV), rabies, papillomavirus, measles, rubella, varicella, rotavirus, polio, variscella zostervirus (VZV), and negative stranded RNA viruses, such as for example, avirus of the family Paramyxoviridae. Examples of a virus of the familyParamyxoviridae include, but are not limited to, human parainfluenzavirus 1, human parainfluenza virus 2, human parainfluenza virus 3, humanparainfluenza virus 4, parainfluenza virus 5, mumps virus, measlesvirus, human metapneumovirus, human respiratory syncytial virus, bovinerespiratory syncytial virus rinderpest virus, canine distemper virus,phocine distemper virus, Newcastle disease virus, avian pneumovirus,Peste des Petits Ruminants virus (PPRV), Sendai virus, Menangle virus,Tupaia paramyxovirus, Tioman virus, Tuhokovirus 1, Tuhokovirus 2,Tuhokovirus 3, Hendravirus, Nipahvirus, Fer-de-Lance virus, Narivavirus, Salem virus, J virus, Mossman virus, and Beilong virus.

An antigenic agent may include one or more immunogens derived frompathogens infectious to poultry. Such immunogens may be derived from,for example, infectious bronchitis virus (IBV), Newcastle disease virus(NDV), Marek's disease (MDV), infectious bursal disease (IBD) virus,infectious laryngotracheitis (ILT), avian reovirus, cholera, fowl pox,mycoplasmosis, turkey and chicken Coryza, avian influenza, avianencephalomyelitis (AE), avian rhinotracheitis (ART), duck virushepatitis, haemorrhagic enteritis, goose parvovirus, Paramyxovirus 3,chicken anaemia virus (CAV), E. coli, Erysipelas, Reimerella, Mycoplasmagallisepticum, Pasteurella multocida, Salmonella enteritidis, Salmonellatyphimurium, coccidiosis, egg drop syndrome (EDS) virus, turkeyrhinotracheitis virus (TRTV), and poxvirus.

An antigenic agent may include one or more immunogens derived frompathogens infectious to bovoids, including, but not limited to, domesticcattle, water buffalo, African buffalo, bison, and yaks. Such immunogensmay be derived from, for example, bovine respiratory disease (BRD)vaccine, including, but not limited to BVDV types I and II, bovineherpes virus 1 (BHV-1) vaccine, including, but not limited to, subunitvaccines that would not result in latent virus, Haemophilus somnusvaccine, Mannheimia haemolytica vaccine, Mycoplasma bovis vaccine,bovine rotavirus vaccine, Escherichia coli K99 vaccine, bovinecoronavirus (BCV) vaccine, Clostridium chauvoei (black leg) vaccine,Clostridium septicum vaccine, Clostridium sordelli (malignant edema)vaccine, Clostridium novyi (black disease) vaccine, Clostridiumperfringens (enterotoxemia) vaccine, infectious bovinekeratoconjunctivitis (pink eye) vaccine, including, but not limited to,Moraxella bovis, chlamydia, mycoplasma, acholeplasma, or infectiousbovine rhinotracheitis (IBR) virus vaccines, mastitis vaccines,including, but not limited to, Escherichia coli J5 vaccine.

An antigenic agent may include one or more immunogens derived frompathogens infectious to swine, including, but are not limited to,porcine circovirus type 2 (PCV2), porcine reproductive and respiratorysyndrome (PRRSV), respiratory mycoplasma, Streptococcus suis, porcinecoronavirus, rotavirus, enterotoxigenic Escherichia coli (K88),Actinobacillus pleuropneumonia (APP), and swine influenza.

An antigenic agent may include one or more immunogens derived from theBurkholderia genus, a group of virtually ubiquitous gram-negative,motile, obligatory aerobic rod-shaped bacteria including bothanimal/human and plant pathogens as well as some environmentallyimportant species. Burkholderia is best known for its pathogenicmembers. Burkholderia mallei is responsible for glanders, a disease thatoccurs mostly in horses and related animals. Burkholderia pseudomalleiis the causative agent of melioidosis (also called Whitmore's disease),an infectious disease predominately of tropical climates that can infecthumans or animals, especially in Southeast Asia and northern Australia.Burkholderia cepacia is an important pathogen of pulmonary infections inpeople with cystic fibrosis. Due to their antibiotic resistance and thehigh mortality rate from their associated diseases Burkholderia malleiand Burkholderia pseudomallei are considered to be potential biologicalwarfare agents, targeting livestock and humans. Burkholderia antigensinclude, for example, any of three burkholderia recombinant proteins(the burkholderia 4-9 protein, the burkholderia 22-11 protein, and theburkholderia 42 protein). Such burkholderia antigens may be administeredseparately or as a cocktail of any two or three antigens.

An antigenic agent may be one or more of those currently used in thecombination measles-mumps-rubella (MMR) andmeasles-mumps-rubella-varicella (MMRV) vaccines.

In some embodiments, the antigenic agent is a polynucleotide vaccine,that is, the antigenic agent is delivered as a vector construct, such asa plasmid, that results in the expression of a polypeptide antigen upondelivery to a subject. As used herein, the term “polynucleotide” refersto a polymeric form of nucleotides of any length, either ribonucleotidesor deoxynucleotides, and includes both double- and single-stranded RNAand DNA. A polynucleotide can be obtained directly from a naturalsource, or can be prepared with the aid of recombinant, enzymatic, orchemical techniques. A polynucleotide can be linear or circular intopology. A polynucleotide may be, for example, a portion of a vector,such as an expression or cloning vector, or a fragment. A polynucleotidemay include nucleotide sequences having different functions, including,for instance, coding regions, and non-coding regions such as regulatoryregions. Any suitable vector or delivery vehicle may be utilized.Various vectors are publicly available. The vector may, for example, bein the form of a plasmid, cosmid, viral particle, or phage. A vector maybe an expression vector. The appropriate nucleic acid sequence may beinserted into the vector by a variety of procedures. In general, DNA isinserted into an appropriate restriction endonuclease site(s) usingtechniques known in the art. Vector components generally include, butare not limited to, one or more of a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence. Construction of suitablevectors containing one or more of these components employs standardligation techniques which are known to those of skill in the art.

A composition as described herein may be useful as a vaccine. Thevaccine may be therapeutic, including, but not limited to, prophylacticor protective.

A composition of the present invention may include one or more compoundswith adjuvant activity. Such an adjuvant stimulates the immune systemand increases the response to a vaccine antigen, without having anyspecific antigenic effect in itself. An adjuvant acts to accelerate,prolong, or enhance antigen-specific immune responses when used incombination with specific vaccine antigens. Suitable compounds orcompositions for this purpose include, but are not limited to, analuminum based adjuvant, such as, for example, aluminum phosphate,aluminum hydroxide (also referred to as alum), aluminumhydroxyl-phosphate, and aluminum hydroxyl-phosphate-sulfate, andnon-aluminum adjuvants, such as, for example, QS21, MF59, Lipid-A,neutral lipsomes, microparticles, a cytokine such as, for example,IL-12, plant oils, animal oils, oil-in-water or water-in-oil emulsionbased on, for example a mineral oil, such as BAYOL F™ or MaARCOL 52™,Complete Freund's adjuvant, incomplete Freund's adjuvant, a vegetableoil such as, for example, vitamin E acetate, a saponin, squalene, alipidated amino acid (“LAA”), and/or a TLR agonist.

In some embodiments, an adjuvant component is a toll-like receptor (TLR)ligand. TLRs in mammals were first identified in 1997. They constitutethe first line of defense against many pathogens and play a crucial rolein the function of the innate immune system. There are many knownsubclasses of Toll-like receptors, including, TLR1, TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, TLR13, TLR14, TLR15,and TLR16, and their ligands exhibit significant structural variation. ATLR agonist is a molecular ligand for one of the various Toll-likereceptors (TLRs). Known TLRs include: TLR1 (TLR1 ligands include triacyllipoproteins); TLR2 (TLR2 ligands include lipoproteins, gram positivepeptidoglycan, lipoteichoic acids, fungi, and viral glycoproteins); TLR3(TLR3 ligands include double-stranded RNA, as found in certain viruses,and poly I:C); TLR4 (TLR4 ligands include lipopolysaccharide and viralglycoproteins); TLR5 (TLR5 ligands include flagellin); TLR6 (TLR6ligands include diacyl lipoproteins); TLR7 (TLR7 ligands include smallsynthetic immune modifiers (such as imiquimod, R-848, loxoribine, andbropirimine) and single-stranded RNA); TLR8 (TLR8 ligands include smallsynthetic compounds and single-stranded RNA); and TLR9 (TLR9 ligandsinclude unmethylated CpG DNA motifs). Some TLR ligands are describedherein, but it should be understood that such listings do not limit theinvention in any way. TLR ligands are widely available commercially.

Preferred TLR agonists include TLR2 agonists, TLR4 agonists, TLR7agonists, TLR8 agonists, and TLR9 agonists. TLR2 is involved in therecognition of a wide array of microbial molecules from Gram-positiveand Gram-negative bacteria, as well as mycoplasma and yeast. TLR2ligands include lipoglycans, lipopolysaccharides, lipoteichoic acids andpeptidoglycans.

TLR4 recognizes Gram-negative lipopolysaccharide (LPS) and lipid A, itstoxic moiety. TLR4 agonists include, but are not limited to,lipopolysaccharide (LPS), viral glycoproteins, monophosphoryl lipid A(MPL) (Anderson et al., 2010, Colloids Surf B Biointerfaces;75(1):123-32), Glucopyranosyl Lipid Adjuvant-Stable Emulsion (GLA-SE)(Coler et al., 2010, PLoS One; 5(10):e13677), and the synthetichexaacylated lipid A derivative, denoted as glucopyranosyl lipidadjuvant (GLA) (Coler et al., 2011, PLoS One; 6(1):e16333). TLR9 isactivated by unmethylated CpG-containing sequences, including thosefound in bacterial DNA or synthetic oligonucleotides (ODNs). Suchunmethylated CpG containing sequences are present at high frequency inbacterial DNA, but are rare in mammalian DNA. Thus, unmethylated CpGsequences distinguish microbial DNA from mammalian DNA. A TLR9 agonistmay be a preparation of microbial DNA, including, but not limited to, E.coli DNA, endotoxin free E. coli DNA, or endotoxin-free bacterial DNAfrom E. coli K12. A TLR9 agonist may be a synthetic oligonucleotidecontaining unmethylated CpG motifs, also referred to herein as “aCpG-oligodeoxynucleotide,” “CpGODNs,” “ODN,” or “CpG.” CpG ODNs areshort, single stranded, DNA molecules that contain a cytosine (“C”nucleotide) followed by a guanine (“G” nucleotide). The “p” typicallyrefers to the phosphodiester backbone of DNA. A TLR9 agonist of thepresent invention may include any of the at least three types ofstimulatory ODNs have been described, type A, type B, and type C.CpG-oligodeoxynucleotides may be produced by standard methods forchemical synthesis of polynucleotides or purchased commercially. Forexample, CPG ODNs can be purchased through InvitroGen (San Diego,Calif.).

The compositions as described herein may include one or more cytokines.Cytokines may include, but are not limited to, IL-1α, IL-1(3, IL-2,IL-3, IL-4, IL-6, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-19,IL-20, IFN-α, IFN-0, IFN-γ, tumor necrosis factor (TNF), transforminggrowth factor-β (TGF-β), granulocyte colony stimulating factor (G-CSF),macrophage colony stimulating factor (M-CSF), granulocyte-macrophagecolony stimulating factor (GM-CSF), and or Flt-3 ligand. In someapplications, one or more cytokines may serve as an adjuvant.

The present invention also includes methods of inducing an immuneresponse in a subject by administering a composition as described hereinto the subject. The immune response may or may not confer protectiveimmunity. An immune response may include, for example, a humoralresponse and/or a cell mediated immune response. A humoral immuneresponse may include an IgG (including IgG1, IgG2 (including IgG2aand/or IgG2b), IgG3, and/or IgG4), IgM, IgA, IgD, IgE, and/or IgYresponse. A cellular immune response may include T cell activationand/or cytokine production. The determination of a humoral or cellularimmune response may be determined by any of a variety of methods knownin the immunological arts, including, but not limited to, any of thosedescribed herein. The induction of an immune response may include thepriming and/or the stimulation of the immune system to a futurechallenge with an infectious agent, providing immunity to futureinfections. The induction of such an immune response may serve as aprotective response, generally resulting in a reduction of the symptoms.The immune response may enhance an innate and/or adaptive immuneresponse. The immune response may demonstrate higher concentrations ofantibodies with a single, primary immunization. The immune response mayshow altered immunoglobulin ratios and/or altered induction ofinflammatory cytokines, type I interferons, and/or chemokines, comparedto immunization without the slurry matrix. Such alteration may be anincrease or a decrease. For example, a higher ratio of one isotype ofimmunoglobulin compared to another immunoglobulin isotype (for example,any one of IgM, IgA, IgD, IgG, or IgE compared to any one of IgM, IgA,IgD, IgG, or IgE) or a higher ratio of one IgG subclass compared toanother IgG subclass (for example, any one of IgG1, IgG2a, IgG2b, IgG3,or IgG4 compared to any one of IgG1, IgG2a, IgG2b, IgG3, or IgG4) may beobtained.

The present invention also includes methods of vaccinating a subject byadministering a composition as described herein to the subject. Suchvaccination may result in a reduction or mitigation of the symptoms offuture infection and may prevent a future infection. The compositionsdescribed herein may have therapeutic and/or prophylactic applicationsas immunogenic compositions in preventing and/or ameliorating infection,such that resistance to new infection will be enhanced and/or theclinical severity of the disease reduced. Such protection may bedemonstrated by either a reduction or lack of the symptoms associatedwith RSS, including, but not limited to, any of those described herein.Any of a wide variety of available assays may be used to determine theeffectiveness of the vaccination method of the present invention,including, but not limited to, any of those described herein.

In some applications, an immunologically effective amount of at leastone immunogen is employed in such amount to cause a substantialreduction in the course of the normal infection. Immunogenicity andeffectiveness may be assayed in any of a variety of known experimentalsystems, including, but not limited to, any of those described herein.

The compositions and methods described herein may be administered to asubject for the treatment and/or prevention of viral diseases,infectious diseases, including, but not limited to bacterial, fungal andparasitic infections, cancer, and other diseases in which theadministration of one or more immunogens is therapeutically desired.With the methods of the present disclosure, the efficacy of theadministration of one or more agents may be assessed by any of a varietyof parameters known in the art, including, but not limited to, any ofthose described herein. This includes, for example, determinations of anincrease in the delayed type hypersensitivity reaction to tumor antigen,determinations of a delay in the time to relapse of the post-treatmentmalignancy, determinations of an increase in relapse-free survival time,determinations of an increase in post-treatment survival, determinationof tumor size, determination of the number of reactive T cells that areactivated upon exposure to the vaccinating antigens by a number ofmethods including ELISPOT, FACS analysis, cytokine release, or T cellproliferation assays.

For example, the compositions and methods described herein may beadministered to a patient for the treatment of cancer. Antitumorvaccines include, but are not limited to, peptide vaccines, whole cellvaccines, genetically modified whole cell vaccines, recombinant proteinvaccines or vaccines based on expression of tumor associated antigens byrecombinant viral vectors. Cancers to be treated include, but are notlimited to, melanoma, basal cell carcinoma, colorectal cancer,pancreatic cancer, breast cancer, prostate cancer, lung cancer(including small-cell lung carcinoma and non-small-cell carcinoma),leukemia, lymphoma, sarcoma, ovarian cancer, Kaposi's sarcoma, Hodgkin'sDisease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,small-cell lung tumors, primary brain tumors, stomach cancer, malignantpancreatic insulanoma, malignant carcinoid, urinary bladder cancer,premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer,neuroblastoma, esophageal cancer, genitourinary tract cancer, malignanthypercalcemia, cervical cancer, endometrial cancer, glioblastoma,adrenal cortical cancer, liver cancer, stomach cancer, oral cancer,cancer of the upper and lower airways, and head and neck. In someaspects, the cancer is a primary cancer. In some aspects, the cancer ismetastatic. As used herein, “tumor” refers to all types of cancers,neoplasms, or malignant tumors found in mammals.

The efficacy of treatment of a cancer may be assessed by any of variousparameters well known in the art. This includes, but is not limited to,determinations of a reduction in tumor size, determinations of theinhibition of the growth, spread, invasiveness, vascularization,angiogenesis, and/or metastasis of a tumor, determinations of theinhibition of the growth, spread, invasiveness and/or vascularization ofany metastatic lesions, determinations of tumor infiltrations by immunesystem cells, and/or determinations of an increased delayed typehypersensitivity reaction to tumor antigen. The efficacy of treatmentmay also be assessed by the determination of a delay in relapse or adelay in tumor progression in the subject or by a determination ofsurvival rate of the subject, for example, an increased survival rate atone or five years post treatment. As used herein, a relapse is thereturn of a tumor or neoplasm after its apparent cessation.

As used herein, unless the context makes clear otherwise, “treatment,”and similar word such as “treated,” “treating,” etc., is an approach forobtaining beneficial or desired results, including and preferablyclinical results. A treatment may include therapeutic and/orprophylactic treatments. Desirable effects of treatment includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of one or more direct or indirect pathological consequencesof the disease, decreasing the rate of disease progression, ameliorationor palliation of the disease state, and remission or improved prognosis.In some applications, a composition as described may demonstrate animprovement in one or more desired results of at least about 10%, atleast about 15%, at least about 20%, or at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, or at least about 95%.

As used herein, an “effective amount” or a “therapeutically effectiveamount” of a substance is that amount sufficient to affect a desiredbiological effect, such as beneficial results, including clinicalresults. Therapeutically effective concentrations and amounts may bedetermined for each application herein empirically by testing thecompounds in known in vitro and in vivo systems, including any of thosedescribed herein. Dosages for humans or other animals may then beextrapolated therefrom. With the methods of the present invention, theefficacy of the administration of one or more interventions may beassessed by any of a variety of parameters well known in the art.

In some embodiments, an “effective amount” is an amount that results ina reduction of at least one pathological parameter. Thus, for example,an amount that is effective to achieve a reduction of at least about10%, at least about 15%, at least about 20%, or at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, or at least about 95%,compared to the expected reduction in the parameter in an individual notreceiving treatment.

The present invention also includes methods of making and using thevaccine compositions described herein. The compositions of the presentdisclosure may be formulated in pharmaceutical preparations in a varietyof forms adapted to the chosen route of administration. In someembodiments, the compositions of the present disclosure may belyophilized.

Any of a variety of modes of administration may be used. For example,administration may be intravenous, topical, oral, intranasal,subcutaneous, intraperitoneal, intramuscular, intratumor, intradermal,mucosal, intrarectal, intravaginal, inhalation, or aerosol.

For aerosol or inhalation delivery, any of a variety of delivery modesmay be used. For example, an inhaler (including, but not limited to,metered dose inhalers and dry powder inhalers), a nebulizer (including,but not limited to air jet and ultrasonic nebulizers), or other aerosoldelivery devise used in the treatment of respiratory illness anddiseases may be used (see, for example, Hess et al., 2007, “A Guide toAerosol Delivery Devices for Respiratory Therapists”). In someembodiments, a commercially available aerosol delivery system used foraerosol delivery to domestic animals and livestock may be used. See, forexample, Palmer et al., 2002, Tuberculosis (Edinb); 82(6):275-82. Insome embodiments, delivery to domestic animals and livestock may be byaerosol spray. Such delivery may target delivery to the mucosal surfacesof the nasal passages and/or the lungs. Any of a variety of particlesizes may be delivered. Particle size may be heterodisperse (also termedpolydisperse), meaning that there is a mix of sizes in the aerosol. Or,particle size may be monodisperse, with particles of a fairly uniformsingle particle size. Particle size plays an important role in lungdeposition. As particle size increases above 3 μm, there is a shift inaerosol deposition from the lung periphery to the conducting airways.Oropharyngeal deposition also increases as particle sizes increase above6 μm. Exhaled loss is high with very small particles of 1 μm or less.Thus, particle sizes of about 1-5 μm may be best for reaching the lungperiphery, while particles of about 5-10 μm deposit preferentially inthe conducting airways. (see Hess et al., 2007, “A Guide to AerosolDelivery Devices for Respiratory Therapists”). For example, particlessize may be selected to target the nose, to target oropharyngealregions, to the conducting airways, to target the upper and centralairways, to target the lower airways, to target the lung periphery, or acombination thereof.

For example, particles may be greater than about 10 μm in diameter,greater than about 6 μm in diameter, greater than about 5 μm indiameter, greater than about 3 μm in diameter, greater than about 2 μmin diameter or greater than about 1 μm in diameter. Particles may about10 in diameter, about 6 μm in diameter, about 5 μm in diameter, about 3μm in diameter, about 2 in diameter, about 1 μm in diameter, or anyrange thereof; for example, about 1 μm to about 10 in diameter, about 1μm to about 6 μm in diameter, about 1 μm to about 5 μm in diameter,about 1 μm to about 3 μm in diameter, about 3 μm to about 5 μm indiameter, about 3 μm to about 6 μm in diameter, about 3 μm to about 10μm in diameter, about 5 μm to about 6 μm in diameter, about 5 μm toabout 10 μm in diameter, or about 6 μm to about 10 μm in diameter.Particles may be less than about 10 μm in diameter, less than about 6 μmin diameter, less than about 5 μm in diameter, less than about 3 μm indiameter, less than about 2 μm in diameter or less than about 1 μm indiameter. Particles may be about 10 μm or less in diameter, about 6 orless in diameter, about 5 μm or less in diameter, about 3 μm or less indiameter, about 2 μm or less in diameter or about 1 μm or less indiameter.

For aerosol delivery, a composition as described herein may be deliveredin a lyophilized, dry, or a liquid state via an aerosolization devise.Once inhaled and encountering physiological pH in the airways and othermucosal sites, the materials would polymerize delivery the vaccineantigen(s), adjuvants, and additional components to the upper and lowerairways.

For oral delivery, a composition as described herein may beadministered, for example, in a capsule, in a liquid drink, orencapsulated in any form of digestible foods or materials. Encounteringphysiological pH in the gut, materials will form biopolymers and deliverthe vaccine antigen(s), adjuvants, and additional components to themucosal immune system.

For vaginal delivery a composition as described herein may beadministered as a spray, wash or douche, or incorporated into a vaginalsponge or vaginal suppository. For intrarectal delivery a composition asdescribed herein may be administered as a spray, wash, or douche, orincorporated into a sponge or rectal suppository. Encounteringphysiological pH in the vagina or rectum, materials will formbiopolymers and deliver the vaccine antigen(s), adjuvants, andadditional components to the mucosal immune system.

A vaccine composition as described herein may take the form of a patchor other vehicle for transdermal delivery. A vaccine composition asdescribed herein may take the form of an implant. Such an implant may beimplanted within the tumor. Delivery may be accomplished by any of avariety of available technologies, including, for example, injection,infusion, instillation, topical application, mucosal application,aerosol delivery, inhalation delivery delivery by a needle, and/ordelivery by a catheter. Delivery may be by the use of a delivery deviceor tool, such as a needle, parch, catheter, or inhalation/aerosoldevise. Such delivery devices are included in the present invention.

A composition of the present invention may be administered with one ormore additional therapeutic interventions. Additional therapeutictreatments include, but are not limited to, surgical resection,radiation therapy, chemotherapy, hormone therapy, anti-tumor vaccines,antibody based therapies, whole body irradiation, bone marrowtransplantation, peripheral blood stem cell transplantation, theadministration of cytokines, antibiotics, antimicrobial agents,antiviral agents, such as AZT, ddI or ddC, the administration ofchemotherapeutic agents, such as, for example, cyclophosphamide,methotrexate, 5-fluorouracil, doxorubicin, vincristine, ifosfamide,cisplatin, gemcitabine, busulfan, ara-C, adriamycin, mitomycin, cytoxan,methotrexate, and combinations thereof. Such administration may takeplace before, during, and/or after the administration of a vaccinecomposition as described.

As used herein, the term “subject” includes, but is not limited to,humans and non-human vertebrates. In some embodiments, a subject is amammal, particularly a human. A subject may be an “individual,”“patient,” or “host.” A subject may include, for example, a human, ahigher primate, a non-human primate, domestic livestock and domesticpets (such as dogs, cats, cattle, horses, pigs, sheep, goats, mules,donkeys, mink, and poultry), laboratory animals (such as for example,mice, rats, hamsters, guinea pigs, and rabbits), and wild life. In someembodiments, a vaccine composition, as described herein is administeredto a bovine, including, but not limited to, domestic cattle, waterbuffalo, African buffalo, bison, and yaks.

The vaccine compositions described herein may be administered topoultry, including, for example, chickens, turkeys, guinea fowl,partridges, and water fowl, such as, for example, ducks and geese.Chickens include, but are not limited to, hens, roosters, broilers,roasters, breeder, the offspring of breeder hens, and layers. Thevaccine of the present invention may be administered to poultry beforeor after hatching. Poultry may receive a vaccine at a variety of ages.For example, broilers may be vaccinated in ovo, at one-day-old, in ovo,or at 2-3 weeks of age. Laying stock or reproduction stock may bevaccinated, for example, at about 6-12 weeks of age and boosted at about16-20 weeks of age. Such laying stock or reproduction stock may bevaccinated at about 6, at about 7, at about 8, at about 9, at about 10,at about 11, or at about 12 weeks of age. Such laying stock orreproduction stock may be boosted at about 16, at about 17, at about 18,at about 19, or at about 20 weeks of age. The offspring of such layingstock or reproduction stock may demonstrate an antibody titer to theadministered immunogen(s), which may prevent or mitigate the symptoms ofan infection in the offspring.

The compositions of the present invention may be formulated according tomethods known and used in the art. A vaccine composition of the presentinvention may include salts, buffers, preservatives, or other substancesdesigned to improve or stabilize the composition. A vaccine compositionmay include a pharmaceutically acceptable excipient or carrier. As usedherein, the term “pharmaceutically acceptable carrier” refers to asubstance suitable for administration to a human or other vertebrateanimal. For administration, a composition as described herein may besuitably buffered if necessary and the composition rendered isotonicwith sufficient saline or glucose. In this connection, sterile aqueousmedia that can be employed will be known to those of skill in the art.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and general safety and purity standards as required by theFDA. Such preparation may be pyrogen-free, may be sterile, and/orendotoxin-free.

A composition of the present invention may also contain one or morestabilizers. Any suitable stabilizer can be used including carbohydratessuch as sorbitol, mannitol, starch, sucrose, dextrin, or glucose;proteins such as albumin or casein; and buffers such as alkaline metalphosphate and the like. A stabilizer is particularly advantageous when adry vaccine preparation is prepared by lyophilization. Such acomposition may include pharmaceutically acceptable carriers ordiluents. Carriers include, for example, stabilizers, preservatives andbuffers. Suitable stabilizers include, for example, SPGA, carbohydrates(such as sorbitol, mannitol, starch, sucrose, dextran, glutamate orglucose), proteins (such as dried milk serum, albumin or casein) ordegradation products thereof. Suitable buffers include, for example,alkali metal phosphates. Suitable preservatives include, for example,thimerosal, merthiolate and gentamicin. Diluents, include, but are notlimited to, water, aqueous buffer (such as buffered saline), alcohols,and polyols (such as glycerol).

Any of a wide variety of modulating agents may be included in themethods and compositions described herein. As used herein, a “modulatingagent” is an agent that has a therapeutic effect on living tissue.Modulatory agents include, for example, therapeutic agents which areeffective to prevent and/or overcome disease and/or promote recovery.

A composition of the present invention may be lyophilized.

The vaccine of the present invention may be administered to a subject byany of many different routes. For example, the vaccine may beadministered intravenously, intraperitonealy, subcutaneously,intranasally, orally, transdermally, intradermally, intramuscularly,intravaginally, intrarectally, and via aerosol for inhalation delivery.Suitable dosing regimes may be determined by taking into account factorswell known in the art including, for example, the age, weight, sex, andmedical condition of the subject; the route of administration; thedesired effect; and the particular conjugate and formulation employed.The vaccine may be administered as either a single does or multipledoses. When administered in a multi-dose vaccination format, the timingof the doses may follow schedules known in the art. For example, afteran initial administration, one or more booster doses may subsequently meadministered to maintain antibody titers and/or immunologic memory.

The methods of the present invention may include in vitro, ex vivo, orin vivo methods. As used herein “in vitro” is in cell culture and “invivo” is within the body of a subject. With the present invention, anisolated immunogen or agent may be delivered. As used herein, “isolated”refers to material that has been either removed from its naturalenvironment, produced using recombinant techniques, or chemically orenzymatically synthesized, and thus is altered “by the hand of man” fromits natural state.

The present invention includes kits employing one or more of thecompositions described herein. Such kits may provide for theadministration of an immunogen to a subject in order to elicit an immuneresponse. Kits of the present invention may include other reagents suchas buffers and solutions needed to practice the invention are alsoincluded. Optionally associated with such container(s) can be a noticeor printed instructions. As used herein, the phrase “packaging material”refers to one or more physical structures used to house the contents ofthe kit. The packaging material is constructed by known methods,preferably to provide a sterile, contaminant-free environment. As usedherein, the term “package” refers to a solid matrix or material such asglass, plastic, paper, foil, and the like, capable of holding withinfixed limits a polypeptide. Kits of the present invention may alsoinclude instructions for use. Instructions for use typically include atangible expression describing the reagent concentration or at least oneassay method parameter, such as the relative amounts of reagent andsample to be admixed, maintenance time periods for reagent/sampleadmixtures, temperature, buffer conditions, and the like.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

The description exemplifies illustrative embodiments. In several placesthroughout the application, guidance is provided through lists ofexamples, which examples can be used in various combinations. In eachinstance, the recited list serves only as a representative group andshould not be interpreted as an exclusive list.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein. All headings are forthe convenience of the reader and should not be used to limit themeaning of the text that follows the heading, unless so specified.

EXAMPLES Example 1 New Vaccination Delivery Regimen Drives Enhanced,Vaccine-Specific Immune Responses

To enhance vaccine delivery over that seen when conventional deliverymethods are used, this example focused on providing antigen, plus orminus CpG adjuvant, along with a component which is in liquid state atroom temperature, but that forms a gel-depot under physiologicalconditions, after injection at 35° C. This allows for the antigen andadjuvant to be delivered in a concentrated form, enhancing antigenpresenting cell activity, and leading to pro-inflammatory,vaccine-specific responses. Recombinant hepatitis B antigen (rHepBag)was used as the antigen for vaccination, and the delivery methodevaluated along with seven different vaccine delivery schemes for theability to induce Hepatitis B specific antibodies and cytokines. Micewere vaccinated with rHepBag in two different types of gel slurries(PURAMATRIX, also referred to herein as “P1,” and MATRIGEL, alsoreferred to herein as “P2”), or with rHepBag in ALHYDROGEL (aluminumsalt) or rHepBAg mixed with Complete Freund's adjuvant (CFA). Gelslurries and ALHYDROGEL were mixed +/− with the murine CpG ODN 1826.

Results showed that mice vaccinated with either gel slurry plus ODN hadsignificantly higher TNF production 24 to 48 hours after primaryinoculation, while P1 was significantly superior to ALHYDROGEL at 24hours. Adjuvant P2 presented a promising Th2 inhibition after 48 hourswith the reduction of IL-4, IL-5 and IL-10 levels coincident withincreased antigen-specific IgG2a production in serum.

The analysis of vaccine-specific antibodies showed that P1 drove highvaccine-specific IgA, IgM and IgG titers 14 days post-prime with orwithout using ODN and the high IgA and IgG titers was maintained for 35days. As both of the gel slurry systems tested in this study wassuperior to the conventional adjuvants, this new gel slurry vaccinedelivery system will have broad utility for enhancing responses tonumerous current vaccines that are currently marginally functional. Theuse of this new vaccine delivery system will be further investigated inthe development of vaccines for any of a wide variety of infectiousdiseases, from parasitic to viral infection.

Material and Methods

Vaccines and Route of Administration. The experimental vaccine used inthis study was produced from a recombinant Hepatitis B antigen, namely,rHepBag (Fitzgerald Industries, Inc. Massachusetts, USA). 90 6- to8-week-old female BALB/c mice were evenly divided into 9 groups andrespectively received a prime subcutaneous injection (sc) on the backand, a boost 4 weeks later, of 0.1 ml solution containing 5 μg ofrHepBag, 0.1 ml of 50 μg ODN 1826 (InvivoGen, Inc. California, USA) with5 μg of rHepBag, 0.4 ml solution of PURAMATRIX (P1) and 5 μg of rHepBagwith or without ODN 1826, 0.4 ml of MATRIGEL (P2) and 5 μg of rHepBagwith or without ODN 1826, 0.1 ml solution of 250 μg alum (Thermo FisherScientific, Inc. Pennsylvania, USA) and 5 μg of rHepBag with or withoutODN 1826 and, 0.1 ml of Complete Freund's Adjuvant (Sigma-Aldrich Co.Missouri, USA) with 5 μg of rHepBag (1:2). To prepare one dose of theslurry, 5 ug of rHBsAg antigen, with or without prior mixing with 50 ugCpG, was brought to a final volume of 400 ul with MATRIGEL or PURAMATRIXand mixed thoroughly before subcutaneous injection. Amounts were scaledup depending on the number of doses needed. MATRIGEL and PURAMATRIX werepurchased from BD (Franklin Lakes, N.J.).

Cytokines and antibody evaluation. Splenocytes were isolated one weekafter boost for cytokines evaluation. Single cell suspensions(1.5×10⁶/ml) were prepared and suspended in 1640 medium (RPMI 1640Thermo Scientific Hyclone, Utah, USA) with penicillin-streptomycin(final concentrations of 100 U/ml and 100 m/ml respectively)(Sigma-Aldrich. St. Louis, Mo., USA). 0.5 ml of the single cellsuspension was added to 48-well plates (Sigma-Aldrich. St. Louis, Mo.,USA) with 0.5 ml of media, 0.5 ml of 1 μg/ml of Concanavalin A (ConA) or0.5 ml of 5 μg/ml of rHepBag and cultured at 37° C. with 5% CO₂. Thelevels of TNF were quantified after 24 and 48 hours culture, IL-4 andIL-5 after 48 hours, IL-4 and IL-10 after 72 hours, each in triplicate.The percentages of cytokine-positive mice were further transformed usinga two-step platform that consisted of (1) to calculate the global medianfor each cytokine considering the whole range of values obtained foreach group; and (2) to establish for each group the concept of ‘low’ and‘high’-cytokine producers using the global median percentage ofcytokine-positive cells as the cut-off edge to segregate the individualsinto two categories named as ‘low’ and ‘high’-cytokine producers. It isimportant to highlight that the overall cytokine profile for each groupwas constructed by giving the same weight to all cytokines and producingcell populations.

IFN gamma ELISpot was also performed with 3×10⁵ and 1.5×10⁵ splenocytesafter 24 hours of culture, as described by the manufacturer (BDBiosciences (San Francisco, Calif., USA) using 1 μg/ml of ConA aspositive control. The spot-forming unit (SFU) value was expressed asmean of the triplicate cultures minus the mean value of its individualbackground.

Blood samples from mice were collected weekly from 1 to 6 weeks,including the day prior to the primary immunization. Sera collected fromthese bleeds were used in ULISA assays for the detection andquantification of antibodies.

Synthetic Peptides for T-cell analysis for flow cytometry. Syntheticpeptides were synthesized by Biosynthesis, Inc., and were selected basedon relevant literature. The S 228-39 peptide (IPQSLDSWWTSL; SEQ ID NO:6)is H2-L^(d)-restricted and the dominant epitope in Balb/c mice. Thesplenocytes from five mice per group were individually stimulated with 5μM peptide and 40 U/ml IL-2 for flow cytometry.

Statistical analyses. For antibody evaluation, comparisons were analyzedby Mann-Whitney or Student's t test using GraphPad PRISM software,version 4.0 (GraphPad Software, California, USA), afterKolmogorov-Smirnov normality test. A difference was considered asstatistically significant when a P-value was <0.05. Chi squared-test wasused for comparisons of ‘low’ and ‘high’-cytokine producers frequenciesamong groups and significance considered at P<0.05. Comparison of radargraphs axes and polygon areas were considered significant for ratios twotimes lower in magnitude. Data analysis for the results presented in theradar chart format was performed by comparing the central polygon areasamong cytokine-producers categories intra and inter groups. Significantdifferences were considered for ratios indicating axes and polygon areastwo times lower or higher in size.

Results

Initial results showed mice vaccinated with either gel slurry plus CpGshad significantly higher vaccine-specific IgG2a 14 days after the prime,and IgA, IgM at 28 days post inoculation than mice vaccinated with alumor CFA. One gel slurry delivery drove significantly highervaccine-specific IgG titers 14 days post-prime than the other deliverymethods did post-boost, suggesting that the boost was unnecessary.Recall assays showed upregulated IL-10 and IL-4 from splenocytes of micevaccinated with ALHYDROGEL or CFA compared to cells from gel-slurry+CpGvaccinated mice. CpG use reduced levels of IL-5 to background in allgroups compared to elevated levels in CFA. No differences in levels ofIFN or TNF were seen.

FIG. 1 shows the kinetics of IgA anti-HBsAg antibody titers. Data shownis pooled from two independent experiments for total n=10.

FIG. 2 shows the kinetics of IgM anti-HBsAg antibody titers. Data shownis pooled from two independent experiments for total n=10.

FIG. 3 shows the kinetics of IgG anti-HBsAg antibody titers. Data shownis pooled from two independent experiments for total n=10.

FIG. 4 shows the kinetics of IgG₁ anti-HBsAg antibody titers. Data shownis pooled from two independent experiments for total n=10.

FIG. 5 shows the kinetics of IgG_(2a) anti-HBsAg antibody titers. Datashown is pooled from two independent experiments for total n=10.

FIG. 6 shows higher anti-HBsAg antibody titers after single vaccination(21 days). Data shown is pooled from two independent experiments fortotal n=10; *p<0.05, **p<0.01, **p<0.001, ****p<0.0001 compared toantigen alone using two-way ANOVA with Bonferroni post-test.

FIG. 7 shows higher anti-HBsAg antibody titers after single vaccination(35 days). Data shown is pooled from two independent experiments fortotal n=10; *p<0.05 compared to antigen alone using two-way ANOVA withBonferroni post-test.

FIG. 8 shows cytokine profile twenty-four hours after HBsAgre-stimulation of splenocytes. Data shown is representative of oneexperiment, n=5; *p<0.05, ****p<0.0001 compared to antigen alone usingtwo-way ANOVA with Bonferroni post-test.

FIG. 9 shows cytokine profile forty-eight hours after HBsAgre-stimulation of splenocytes. Data shown is pooled from two independentexperiments for total n=10 (n=5 for adjuvants only); no statisticaldifferences seen (p<0.05) compared to antigen alone using two-way ANOVAwith Bonferroni post-test.

FIG. 10 shows cytokine profile seventy-two hours after HBsAgre-stimulation of splenocytes. Data shown is pooled from two independentexperiments for total n=10 (n=5 for adjuvants only); **p<0.01 comparedto antigen alone using two-way ANOVA with Bonferroni post-test.

FIG. 11 shows increased HBsAg-specific cell-mediated immunity by ELISpotin HbsAg-specific T cell responses. Data shown is pooled from twoindependent experiments for total n=10; *p<0.05 compared to antigenalone using two-way ANOVA with Bonferroni post-test.

FIG. 12 shows increased HBsAg-specific T cell-mediated immunity by flowcytometry. Data shown is pooled from two independent experiments fortotal n=10; **p<0.01 compared to antigen alone using two-way ANOVA withBonferroni post-test.

Increase of cytokines secretion by splenocytes with stimulation ofrHepBag. In order to measure the cellular immune response induced by thevaccines, mice were sacrificed one week after boost and splenocytes werecultured with or without 5 μg/ml final concentration of rHepBag.Cytokines levels were measured by ELISA. The concept of low and highproducers was applied to investigate a broader range of cytokines andassess ex vivo cytokine profiles of circulating leukocytes. For thispurpose, the global median for each cytokine-positive cell subset wascalculated, taking the whole range of values obtained for each group, asdescribed elsewhere (Vitelli-Avelar et al., 2008, Scand J Immunol;68(5):516-25). The global median percentage of each cytokine-positivecell population was used as the cut-off edge to segregate theindividuals into two categories named ‘low’ and ‘high’-cytokineproducers, as shown in Table 1.

The data demonstrated that none of the mice inoculated with ODN 1826showed TNF production in 24 hours, but when ODN 1826 was associated withadjuvants P1 or P2, a significant proportion of mice displayed values ofTNF above the cut-off edge, as shown in FIG. 13 (P<0.011). AlthoughALHYDROGEL, when associated or not to ODN 1826, induced a significantamount of ‘high’-TNF producers, all of the mice inoculated with adjuvantP1 without CpG displayed values above the cut-off, and this value wassignificantly higher than all Alhydrogel groups (P<0.001). This sameresult was not observed after 48 hours when adjuvants P1, P2, ALHYDROGELand Freund showed a comparable number of ‘high’-TNF producers (FIGS. 14Aand 14B) (P<0.001).

Moreover, interesting data was found for IL-4 and IL-5 production after48 hours when most of the mice immunized with adjuvant P2 fell into aregion of ‘low’-IL-4 and IL-5 producers (P<0.05). Differently, most ofthe mice inoculated with adjuvant P1 presented a high production of bothcytokines (P<0.002). As shown in FIG. 15, the adjuvant P1 continued topresent ‘high’-IL-4 producers after 72 hours, in association or not withODN 1826 and this same result was found after ALHYDROGEL or Freundimmunization (P<0.001). Also, the data demonstrated that most of themice previously immunized with adjuvant P2 or adjuvant P2 plus ODN 1826displayed values of IL-10 below the cut-off edge, significantly lowerthan mice immunized with rHepBag, associated or not to ODN 1826(P<0.05).

Sustained humoral response in mice vaccinated with rHepBag and adjuvantsP1 and P2. Sera were collected weekly before and after immunization totest specific isotypes antibody titer by ELISA. Mice vaccinated withboth adjuvants P1 and P2 developed an anti-rHepBag IgG antibody in 2weeks after prime inoculation (P<0.002). The highest anti-rHepBagantibody titers were reached in mice vaccinated with P1 associated ornot to ODN, and these responses were significantly greater thanimmunization with ODN, Alum or Freund's adjuvants (P<0.001). TheIgG1:IgG2a ratio elicited in vivo represents different patterns for bothadjuvants. Adjuvant P1 drives to a Th2 response with high levels of IgG1in 14 to 35 days post-prime inoculation. Whereas, the response elicitedby P2 plus ODN is a mixed system rather than a pure Th1 or Th2 response,wherein the IgG1 and IgG2a levels were upregulated during all thetimeline. See FIG. 17A-17D.

The combination of adjuvant P1+/−ODN induced the upregulation of IgA andIgM titers as soon as 14 days post-prime inoculation and this humoralresponse was maintained until 35 and 21 days, respectively (P<0.04).This adjuvant was superior to Freund's during 14 and 35 days and toALHYDROGEL for the first 3 weeks for the production of IgA, IgM and IgG(P<0.02). Additionally, both adjuvants P1 and P2 demonstrated to besuperior to Freund's adjuvant for the production of all Ig after boost(P<0.02). See FIGS. 16A-16D.

Discussion

This example showed that mice vaccinated with either a P1 or P2 gelslurry plus ODN had significantly higher TNF production 24 to 48 hoursafter primary inoculation, while P1 was significantly superior toALHYDROGEL at 24 hours. Adjuvant P2 presented a promising Th2 inhibitionafter 48 hours with the reduction of IL-4, IL-5 and IL-10 levelscoincident with increased antigen-specific IgG2a production in serum.

The analysis of vaccine-specific antibodies showed that P1 drove highvaccine-specific IgA, IgM and IgG titers 14 days post-prime with orwithout using ODN and the high IgA and IgG titers was maintained for 35days. As both of the gel slurry systems tested in this study weresuperior to the conventional adjuvants, this new gel slurry vaccinedelivery system will have broad utility for enhancing responses tonumerous current vaccines that are currently marginally functional. Theuse of this new vaccine delivery system will be further investigated inthe development of vaccines for any kind of infectious diseases, fromparasitic to viral infection.

TABLE 1 Frequency of cytokine high-producers subjects based on theglobal median cytokine cut-off detected in splenocytes culturestimulated with rHepBag* High cytokines producers (%) rHepBag rHepBagrHepBag in Global median rHepBag + rHepBag in P2 + rHepBag in P1 +rHepBag in Alhydrogel + rHepBag Cytokine cut off rHepBag ODN in P2 ODNin P1 ODN Alhydrogel ODN in Freund 24 h TNF  0.009 40 0 40  25^(b)  100^(a, c, d)  40^(b) 75^(a)  80^(b) 40  (0.00-1.26) 48 h TNF 12.96 20 40  50^(a)  56^(b) 50^(a) 30 67^(a)  60^(b) 80^(a)  (0.73-16.41) IFNgamma 0.00 50 50 50 56 40  60 33  30 20  (0.00-3.89) IL-4 0.00 20 0 10^(a)  0 40^(a)  0 33^(a)  0 50^(a) (0.00-0.00) IL-5 0.00 40 10 20^(a)  0 80^(a)  0 67^(a)  0 80^(a) (0.00-5.98) 72 h IL-4 0.00 40 10 30  0  90^(a, e)  30^(b) 89^(a)  0 80^(a) (0.00-7.23) IL-10 41.57  7040 50^(a)  11^(b) 80  30 56  30 80   (0.00-179.00) *Data are expressedas percentage of mice displaying percentage of cytokine⁺ cells higher orequal the global median cut-off calculated for each cell populationwithin the adaptive immunity cells. Statistical significance at P ≤ 0.05(x²) are represented by superscript letters ‘a’, ‘b’, ‘c’, ‘d’ and ‘e’for comparisons with rHepBag, rHepBag + ODN, rHepBag in Alhydrogel,rHepBag in Alhydrogel + ODN and rHepBag in Freund, respectively.PURAMATRIX (P1) and MATRIGEL (P2).

Example 2 Influenza Vaccination

Following methods described in more detail in the previous examples,C57/BL and Balb/c mice were immunized with the recombinant nucleoprotein(rNP) influenza virus antigen administered with alum, CpG, or a slurryof PURAMATRIX and CpG. To prepare one dose of the slurry, 10 ug rNPantigen, with or without prior mixing with 50 ug CpG, was brought to afinal volume of 200 ul with PURAMATRIX and mixed thoroughly beforesubcutaneous injection. Amounts were scaled up depending on the numberof doses needed. As shown in FIG. 18B, anti-influenza IgG titers wereincreased Balb/c mice vaccinated with rNP administered as a slurry withPURAMATRIX and CpG, as compared to mice vaccinated with rNP administeredwith the adjuvant alum or CpG. FIG. 18A shows anti-influenza IgG titersin C57BL/6 mice. Anti-influenza IgG titers were determined four weekspost-last vaccination (wplv).

Again, following methods described in more detail in the previousexamples, C57Bl/6 mice were immunized with whole inactivated influenza Avirus (H1N1) strain PR8 administered with alum, as a PURAMATRIX slurry,or as a slurry of PURAMATRIX and CpG. As a control, additional mice wereimmunized with PR8 whole inactivated virus (WIV) only. To prepare onedose of the slurry, 15 ug of PR8 antigen, with or without prior mixingwith 50 ug CpG, was brought to a final volume of 200 ul with PURAMATRIXand mixed thoroughly before subcutaneous injection. Amounts were scaledup depending on the number of doses needed. PR8 (WIV) isformalin-inactivated influenza A/PR/8/34 (H1N1) from Charles River rNPis recombinant human Influenza A (A/PR/8/34/Mount Sinai (H1N1) segment5) nuclear protein NP from Imgenex.

As shown in FIG. 19, serum anti-influenza IgG titers were increased inC57Bl/6 mice vaccinated with PR8 WIV administered as a slurry ofPURAMATRIX and CpG, as compared to mice vaccinated with PR8 WIVadministered with the adjuvant alum or as a slurry with PURAMATRIXalone, without CpG. Anti-influenza IgG titers were determined four weekspost-last vaccination (wplv). FIG. 19 shows results from two independentexperiments.

As show in FIG. 20, enhanced protection from lethal challenge wasobserved in C57Bl/6 mice vaccinated with PR8 WIV. FIG. 20A shows resultsfrom an independent experiment with lethal challenge at 30 LD₅₀ and FIG.20B shows results from an independent experiment with lethal challengeat 1000 LD₅₀.

Example 3 Burkholderia Vaccination

Following methods described in more detail in the previous example, micewere immunized with a cocktail of three different burkholderiarecombinant proteins (burkholderia 4-9 protein, the burkholderia 22-11protein, and the burkholderia 42 protein) administered with one of threedifferent adjuvants preparation, alum, Complete Freund's Adjuvant (CFA),or slurry of PURAMATRIX and CpG. To prepare one dose of thePURAMATRIX+CpG slurry, 75 ug of Burkholderia proteins antigen, with orwithout prior mixing with 50 ug CpG, was brought to a final volume of200 ul PURAMATRIX and mixed thoroughly before subcutaneous injection.Amounts were scaled up depending on the number of doses needed. Ascontrols, additional mice were immunized with alum only, CFA only, orPURAMATRIX+CpG slurry, without antigen.

The Burkholderia (previously part of Pseudomonas) genus name refers to agroup of virtually ubiquitous gram-negative, motile, obligatory aerobicrod-shaped bacteria including both animal/human and plant pathogens aswell as some environmentally important species. Burkholderia is bestknown for its pathogenic members. Burkholderia mallei is responsible forglanders, a disease that occurs mostly in horses and related animals.Burkholderia pseudomallei is the causative agent of melioidosis (alsocalled Whitmore's disease), an infectious disease predominately oftropical climates that can infect humans or animals, especially inSoutheast Asia and northern Australia. Burkholderia cepacia is animportant pathogen of pulmonary infections in people with cysticfibrosis. Due to their antibiotic resistance and the high mortality ratefrom their associated diseases Burkholderia mallei and Burkholderiapseudomallei are considered to be potential biological warfare agents,targeting livestock and humans.

FIG. 21 shows anti-burkholderia IgG titers in mice immunized with acocktail of three burkholderia recombinant proteins (burkholderia 4-9protein, the burkholderia 22-11 protein, and the burkholderia 42protein). FIG. 21B shows anti-burkholderia protein 4-9 IgG titers inimmunized mice. FIG. 21A shows anti-burkholderia protein 22-11 IgGtiters in immunized mice. FIG. 21C shows anti-burkholderia protein 42IgG titers in immunized mice. Antibody titers after immunization withalum only, alum+protein cocktail, complete Freund's adjuvant (CFA) only,CFA+protein cocktail, PURAMATRIX+CpG slurry only, and PURAMATRIXgel+protein cocktail are shown. An increased immune response as measuredby specific serum antibody levels were observed against two of the threeproteins administered as a gel vaccine composition with puramatrixcompared with vaccination with alum or CFA. Specifically, increasedanti-burkholderia IgG titers were observed against the burkholderia 4-9protein (FIG. 21B) and the burkholderia 22-11 protein (FIG. 21AB) whenadministered as a gel vaccine with PURAMATRIX, compared with vaccinationwith alum or CFA. Challenge data is being analyzed.

Example 4 Veterinary Vaccines

Following procedures described in more detail in the previous examples,the present invention may be used with any of a variety of veterinaryvaccines. The vaccine compositions, delivery methods, and deliverysystem of the present invention will provide many advantages and bettervalue for the commercial livestock industry, including, but not limitedto, improved efficacy, delivery early in the production cycle, andefficacy with the administration of only a single dose to provideprotection throughout the production cycle.

The compositions, delivery methods, and delivery systems of the presentinvention may be used in the immunization of swine. Vaccines that may beadministered to swine using the compositions, methods and systems of thepresent invention include, but are not limited to, porcine circovirustype 2 (PCV2) vaccine, porcine reproductive and respiratory syndrome(PRRSV), respiratory mycoplasma vaccine, Streptococcus suis vaccine,porcine coronavirus vaccine, rotavirus vaccine, enterotoxigenicEscherichia coli (K88) vaccine, Actinobacillus pleuropneumonia (APP)vaccine, and swine influenza vaccine. See, for example, the world wideweb at merck-animal-health.com/species/pigs/vaccines.aspx, “Vaccinationsfor the Swine Herd,” Alabama Cooperative Extension System PublicationANR-902, Alabama A&M and Auburn Universities (available on the worldwide web at aces.edu/pubs/docs/A/ANR-0902/ANR-0902.pdf), and “Pigvaccination programs,” PRIME FACT publication 944, September 2009(available on the world wide web at dpi.nsw.gov.au/_data/assets/pdffile/0009/301500/Pig-vaccination-programs.pdf) for more detailedinformation on available vaccines and the administration of suchvaccines to swine.

The compositions, delivery methods, and delivery systems of the presentinvention may be used in the immunization of bovine, including, but notlimited to, domestic cattle, water buffalo, African buffalo, bison, andyaks. Vaccines that may be administered to bovines, using thecompositions, methods and systems of the present invention include, butare not limited to, bovine respiratory disease (BRD) vaccine, including,but not limited to BVDV types I and II, bovine herpes virus 1 (BHV-1)vaccine, including, but not limited to, subunit vaccines that would notresult in latent virus, Haemophilus somnus vaccine, Mannheimiahaemolytica vaccine, Mycoplasma bovis vaccine, bovine rotavirus vaccine,Escherichia coli K99 vaccine, bovine coronavirus (BCV) vaccine,Clostridium chauvoei (black leg) vaccine, Clostridium septicum vaccine,Clostridium sordelli (malignant edema) vaccine, Clostridium novyi (blackdisease) vaccine, Clostridium perfringens (enterotoxemia) vaccine,infectious bovine keratoconjunctivitis (pink eye) vaccine, including,but not limited to, Moraxella bovis, chlamydia, mycoplasma,acholeplasma, or infectious bovine rhinotracheitis (IBR) virus vaccines,mastitis vaccines, including, but not limited to, Escherichia coli J5vaccine.

In some applications, the compositions, delivery methods, and deliverysystems of the present invention may be used for the administration ofone or more schistosomiasis antigens to a bovoid. Such a schistosomeantigen may be derived from, for example, Schistosoma japonicum,Schistosoma monsoni, or Schistosoma haematobium. A schistosome antigenmay be a schistosome triose phosphate isomerase (CTPI) protein, orantigenic fragment or derivative thereof, including, but not limited toa S. japonicum, S. monsoni, or S. haematobium CTPI protein, or antigenicfragment or derivative thereof. A schistosome antigen may be aschistosome tetraspin 23 kDa integral membrane protein (C23), orantigenic fragment or derivative thereof, including, but not limited toa S. japonicum, S. monsoni, or S. haematobium C23 protein, or antigenicfragment or derivative thereof. Such a schistosome antigen may be achimeric polypeptide, fused to one or more additional antigenicdeterminants, such as for example, a heat shock protein, or antigenicfragment or derivative thereof, including, but not limited to, bovineheat shock protein 70 (Hsp70).

See, for example, “Beef Cattle Herd Health Vaccination Schedule” Powellet al., University of Arkansas, Division of Agriculture, Agriculture andNatural Resources publication FSA3009 (available on the world wide webat uaex.edu/Other Areas/publications/PDF/FSA-3009.pdf), “How toVaccinate,” Oklahoma Cooperative Extension Service, Division ofAgricultural Sciences and Natural Resources, Publication No. 350(available on the world wide web at ansci.colostate.edu/pdffiles/YLE/Dairy7 vaccinate.pdf), and “Cattle Vaccines and Their Use,”Beef Cattle Handbook publication BCH-3015 (available on the world wideweb at iowabeefcenter.org/Beef %20Cattle %20Handbook/VaccinesCattle.pdf) for more detailed information on available vaccines and theadministration of such vaccines to cattle.

The vaccine compositions, delivery methods, and delivery system of thepresent invention may also be used in the vaccination of companionanimals, including, but not limited to cats and dogs, such as, forexample, for the immunization of dogs with parvovirus vaccine fortransfer of maternal immunity.

Routes of administration include, but are not limited to, subcutaneous(sc) or intramuscular (im) injection. Vaccination with the composition,methods, and delivery systems of the present invention will yield rapid,longer lasting protection after administration of a single dose.

Example 5 Poultry Vaccines

Following procedures described in more detail in the previous examples,the present invention may be used as a delivery system for any of avariety of poultry vaccines, including, but not limited to, vaccines forinfectious bronchitis (IB), Newcastle disease (ND), Marek's diseases,infectious bursal disease (IBD) virus, infectious laryngotracheitis(ILT), avian reovirus, cholera, fowl pox, mycoplasmosis, turkey andchicken Coryza, avian influenza, avian encephalomyelitis (AE), avianrhinotracheitis (ART), duck virus hepatitis, haemorrhagic enteritis,goose parvovirus, Paramyxovirus 3, chicken anaemia virus (CAV), E. coli,Erysipelas, Reimerella, Mycoplasma gallisepticum, Pasteurella multocida,Salmonella enteritidis, Salmonella typhimurium, and coccidiosis.

Routes of administration include, but are not limited to, subcutaneous(sc) or intramuscular (im) injection. With subcutaneous injection,vaccine is injected into the space between the skin and underlyingtissues. Typically the site of application used in poultry in the looseskin at the back of the neck. With intramuscular injection, the vaccinepreparation is deposited within a mass of muscle. Typically, either thebreast muscle or the thigh muscle is used for this purpose. Vaccinationmay include the vaccination of day old hatchlings, pullets, layers,breeders, broilers, and/or show birds. Typically layers and breeders getvaccinated prior to the laying period, and then every 6 to 8 weeks withinactivated vaccines during the laying period. This not only protectsthem from disease but passes on maternal antibodies to the progeny.Poultry that may be vaccinated include, but are not limited to,chickens, turkeys, and waterfowl, such as, for example, ducks and geese.

Example 6 Schistosomiasis Immunization

Mice were immunized with the schistosome protein antigen CCA in eithercomplete Freund's adjuvant or in MATRIGEL plus CpGs. Anti-CCA IgGantibody titers were determined by ELISA The data is shown in FIG. 22A(CCA in complete Freund's adjuvant) and FIG. 22B (CCA in MATRIGEL plusCpGs). Two mice were immunized for each antigen preparation. Serasamples were collected at 0, 8, 16, and 19 days post immunization formice immunized with CCA in complete Freund's adjuvant and at 0, 8, 16,24, 32, and 40 days post immunization for mice immunized with CCA inMATRIGEL plus CpGs. The ELISA data clearly shows higher titers of CCAspecific antibodies in mice immunized with MATRIGEL plus CpGs.

Example 7 Schistosomiasis Vaccination of Water Buffalo

Schistosomiasis is a parasitic disease affecting more than 200 millionpeople worldwide. Reassessment of schistosomiasis-related disability,combined with recent information on the global prevalence of schistosomeinfection indicates that the true burden of schistosomiasis issubstantially greater than previously appreciated. In Asia, particularlyChina, the causative agent is Schistosoma japonicum. Unlike the Africanspecies, S. mansoni and S. haematobium, S. japonicum is a zoonoticparasite, with bovines, particularly water buffaloes accounting forabout 75% of schistosome transmission to humans in China. Interventionsthat reduce schistosome infection in water buffaloes will enhance theirhealth simultaneously reducing disease transmission to humans. Currentcontrol programs in many areas of China include simultaneouspraziquantel (PZQ) treatment of humans and water buffaloes; while thishas shown a reduction in the overall prevalence, it requires continuedmass treatments that are both time consuming and expensive. A moresustainable option would be development of a vaccine which reducestransmission of S. japonicum from bovines to replace bovinechemotherapy. Indeed mathematical modeling (Williams et al., 2002, ActaTrop; 82(2):253-262) has demonstrated that reducing S. japonicuminfection in bovine reservoirs using prophylactic vaccines with 45%efficacy alone or in combination with PZQ should over time reduce theequilibrium prevalence and potentially lead to long-term sustainablecontrol of schistosomiasis. This two-pronged base intervention wouldsignificantly reduce transmission of schistosomiasis for the long term,increase bovine health and growth and would likely reduce overallmorbidity in village populations. See Da′Dara et al., 2008, Vaccine;26(29-30):3617-3625, which is herein incorporated by reference in itsentirety.

Antigen-PURAMATRIX compositions as described herein will be used toimmunize livestock with S. japonicum antigens. For these trials, allanimals will be given a primary vaccination with a SjCTPI-Hsp70 plasmidDNA vaccine (as described in more detail in Da′Dara et al., 2008,Vaccine; 26(29-30):3617-3625). Water buffalo will be boosted with acomposition of recombinant SjCTPI protein, PURAMATRIX and bovine CpG.Specifically, 100 ug of recombinant SjCTPI plus bovine CpG will be mixedin PURAMATRIX for a total injection volume of approximately 0.50ml/animal. This will be injected into the shoulder of buffalo/andcattle. Only a single booster vaccination will be administered to ananimal.

The elicitation of a humoral and cellular immune responses, includinganti-SjCTPI IgG antibody response, will be determined. After boosting,animals will be challenged were challenged with cercariae and vaccineefficacy determined by measuring the reduction in the number of eggs pergram feces, reduction in eggs in liver tissues, reduction in miracidialhatching, and reduction in worm burden. Methods for these determinationsare described in more detail in Da′Dara et al., 2008, Vaccine;26(29-30):3617-3625.

A first trial in the Philippines is already in Year 2 in thePhilippines, with 400 water buffalo or cattle having been boosted, asdescribed above. A second trial in Samar, Philippines, will include 1500water buffalo or cattle. A third trail in China will include 600 waterbuffalo or cattle.

Immunization of water buffalo and cattle with compositions ofschistosome polypeptides, such as for example, SjCTPI, SjCTPI-Hsp70,SjC23, or SjC23-Hsp70 polypeptides, in a puramatrix composition, with orwithout adjuvants, such as for example, CpG or IL-12, may serve as thebasis for new control programs for schistosomiasis in Asia. Such aprogram, in addition to treatment with praziquantel (PZQ), would includevaccination of water buffaloes with partially protective vaccines suchSjC23-Hsp70 and SjCTPI-Hsp70 as a means to reduce numbers of egg-layingparasites in livestock, leading to measurable declines in prevalence,intensity and transmission of S. japonicum.

Example 8 New Vaccination Delivery System Provides IncreasedInfluenza-Specific Antibody Response After Primary Immunization andBoost

The preparation of vaccine compositions is described in more detail inthe previous examples. BALB/C mice and C57BL/6 mice were immunized bysub-cutaneous injection with recombinant nucleoprotein (rNP) influenzavirus antigen administered with alum, CpG, or a slurry of PURAMATRIX(“X”) and CpG. As a control, additional mice were given a naïve (mock)injection. A schematic of the method is shown in FIG. 23A. A bloodsample from mice was collected the day prior to immunization. Asub-cutaneous boost was given at week 3, another blood sample from micewas collected at 6 weeks, and mice were harvested at week 7.

As shown in FIG. 23, serum anti-influenza IgG titers were increased inBALB/C mice (FIG. 23B) vaccinated with α-NP administered with as aslurry of PURAMATRIX and CpG, as compared to mice vaccinated with α-NPadministered with the adjuvant alum or as a slurry with PURAMATRIXalone, without CpG. Similarly, anti-influenza IgG titers were slightlyincreased in C57BL/6 mice (FIG. 23C) vaccinated with α-NP administeredwith as a slurry of PURAMATRIX and CpG, as compared to mice vaccinatedwith α-NP administered with the adjuvant alum and were significantlyincreased as compared to mice vaccination with a slurry with PURAMATRIXalone, without CpG. Anti-influenza IgG titers were determined six weekspost-vaccination (wpv).

An increased humoral response was observed in C57BL/6 mice vaccinatedwith rNP via the new delivery method and further studies with theInfluenza model were pursued as described in the following examples.

Example 9 New Vaccination Delivery System Provides IncreasedInfluenza-Specific Antibody Response and Increased Protection fromChallenge After Single Immunization

The preparation of vaccine compositions is described in more detail inthe previous examples. In an effort to mimic the seasonal vaccinereceived by the high-risk groups, a single vaccination of inactivatedvirus was used. A whole-inactivated influenza A virus (WIV) strainPuerto Rico/08/34 (PR8) is considered the most immunogenic and was usedin this study. The humoral response to the vaccine is often consideredthe most important element for protection and is therefore the focus ofthis study.

C57BL/6 mice were immunized with PR8 WIV administered with alum, withCpG, as a PURAMATRIX slurry, or as a slurry of PURAMATRIX and CpG. Ascontrols, additional mice were immunized with PR8 whole inactivatedvirus (WIV) only or were given a naïve (mock) injection. A schematic ofthe method is shown in FIG. 24A. Blood samples from mice were collectedthe day prior to immunization and at weeks 2 and 4. A lethal influenzachallenge of live homologous PR8 at 1,000 LD₅₀ was given by intra-nasal(i.n.) administration at week 4, and mice were harvested at week 6. Micewere monitored daily from challenge until harvest and were analyzed forboth specific antibody titers and morbidity and mortality results.

Influenza-specific IgG endpoint titers were determined from sera samplescollected 4 weeks post single vaccination and immediately prior tochallenge. As shown in FIG. 24, a 2-fold increase in α-PR8influenza-specific antibody induction is seen in mice after a singlevaccination with PR8 administered with as a slurry of PURAMATRIX andCpG, as compared to mice vaccinated with PR8 administered with CpG, micevaccinated with PR8 administered with the adjuvant alum, or micevaccinated with PR8 alone (FIG. 24B). In addition, a significantincrease in influenza-specific antibody induction is seen in mice aftera single vaccination with PR8 administered with as a slurry ofPURAMATRIX and CpG, as compared to mice vaccinated with PR8 administeredas a slurry of PURAMATRIX alone, without CpG. FIGS. 24B and 24C arerepresentative of one triplicate experiment (n=5-9). FIG. 25 showsadditional analysis of influenza-specific IgG endpoint titers determinedfrom sera samples collected 4 weeks post last vaccination. As shown ineach of FIGS. 25A and 25B, an increase in α-PR8 influenza-specificantibody induction is seen in mice after a single vaccination with PR8administered with as a slurry of PURAMATRIX and CpG, as compared to micevaccinated with PR8 administered with CpG, mice vaccinated with PR8administered with the adjuvant alum, mice vaccinated with PR8 alone, ormice given a control (naïve) injection. FIGS. 25A and 25B arerepresentative of one triplicate experiment (n=5-9).

To further characterize this increase in influenza-specific antibodies,isotype and subtype analyses were performed. Influenza-specific IgE,IgA, IgM and IgG antibodies for different vaccine groups were analyzed.As shown in FIG. 26, an α-PR8 antibody class analysis of pre-challengesera (at 4 weeks post vaccination) demonstrated increased endpointtiters of IgE (FIG. 26A), IgA (FIG. 26B), IgM (FIG. 26C), and IgG(10-fold; FIG. 26D) in mice after a single vaccination with PR8administered with as a slurry of PURAMATRIX and CpG.

IgG-subtypes were further examined to determine the type of response(Th1 or Th2). Influenza-specific IgG (λ1), IgG(2a), IgG(2b) and IgG(3)levels for the same 6 vaccine groups were analyzed. As shown in FIG. 27,an IgG antibody subtype analysis of pre-challenge sera (at 4 weeks postvaccination) demonstrated increased endpoint titers of IgG-λ-1 (FIG.27A), IgG-2a (FIG. 27B), IgG-2b (FIG. 27C), and IgG-3 (FIG. 27D) in miceafter a single vaccination with PR8 administered with as a slurry ofPURAMATRIX and CpG. The high endpoint titers & significant increases inthe new method, over the other vaccine groups, in IgG(1) and IgG(2b)suggests a mixed Th1/Th2 response.

Morbidity and mortality were examined to determine whether this enhancedhumoral response would correlation to protection. Following samevaccination schedule and groups described previously, mice werechallenged a 4 weeks post vaccination with 1000LD₅₀ PR8. Morbidity wasdetermined by daily monitoring and is expressed as the mean percentweight (FIG. 28A) change and the mean body score (FIG. 28B), for eachgroup, over time post challenge. Body scores are comprised of severalfactors including weight loss, behavior and physical appearance. Thus,body score is a quantitative measure of symptom severity. As shown inFIG. 28, morbidity and mortality are decreased following lethal,homologous flu challenge at 1,000 LD₅₀. Mice that received a singlevaccination of PR8 administered with as a slurry of PURAMATRIX and CpG,PR8 administered with CpG, PR8 administered with the adjuvant alum, orPR8 WIV maintained body weight (FIG. 28A) and body score(FIG. 28B). ThePR8+X group was not included in this challenge; however, the PR8+X groupwas compared to these groups in another experiment and had a similartrend as the naïve group. These results indicate that the new method,has a decreasing effect on morbidity Mortality was determined by lookingat the percent survival over time post challenge, for each group. Weobserved that the new method is the only group able to provide 100%protection against a 1,000LD₅₀ PR8 challenge (FIG. 28C). Multiplerepeats show the new delivery method consistently decreasing morbidityand mortality rates following lethal PR8 challenge.

Example 10 Enhanced Viral Clearance Following Vaccination

The preparation of vaccine compositions is described in more detail inthe previous examples. Mice were immunized with whole inactivatedinfluenza A virus (H1N1) strain Puerto Rico/08/34 (PR8) wholeinactivated virus (WIV) or were immunized with PR8 administered as aslurry of PURAMATRIX and CpG. As a control, additional mice wereimmunized given a naïve (mock) injection. A schematic of the method isshown in FIG. 29A. Blood samples from mice were collected the day priorto immunization and at week 4. A lethal influenza challenge of livehomologous PR8 at 1,000 LD₅₀ was given by intra-nasal (in)administration at week 4. A sample of mice from each group wassacrificed at various time points post challenge (e.g., at 1, 2, 3, and5 days post challenge) and the lungs were analyzed for viral clearance.Plaque assays were used to determine the amount of virus present in thelungs.

As shown in FIG. 29B, the amount of plaque forming units per lung ofvaccinated mice decreased in the later time points as they were able toclear virus. Mice given a naïve injection received replicating virus andhad high levels of plaque forming units across all time points. Miceimmunized with PR8 WIV show day 1 and day 2 virus levels approximately 2logs lower than mice in the naïve group and were clearing virus by day 5post challenge. Mice immunized with PR8 administered as a slurry ofPURAMATRIX and CpG had significantly lower viral burdens across all timepoints. At 1 day post challenge plaque forming units were almost 2 logslower than the naïve group. At 2 days post challenge plaque formingunits were almost 4 logs lower than the naïve group and at approximately2 logs lower than the mice vaccinated with PR8 WIV.

FIG. 30 shows additional analysis of influenza-specific IgG endpointtiters determined from sera samples collected 1 day post challenge. Asshown in each of FIGS. 30A and 30B, an increase in α-PR8influenza-specific antibody induction is seen at 1 day post challenge inmice vaccinated with PR8 alone and in mice vaccinated with PR8administered with as a slurry of PURAMATRIX and CpG, as compared to micegiven a control (naïve) injection.

FIG. 31 shows additional analysis of influenza-specific IgA endpointtiters determined from sera samples collected 1 day post challenge. Asshown in each of FIGS. 31A and 31B, an increase in α-PR8influenza-specific antibody induction is seen at 1 day post challenge inmice vaccinated with PR8 administered with as a slurry of PURAMATRIX andCpG, as compared to mice vaccinated with PR8 alone. An increase in α-PR8influenza-specific antibody induction is seen at 1 day post challenge inmice vaccinated with PR8 alone when compared to mice given a control(naïve) injection.

Example 11 Inter-Strain Reactivity

The preparation of vaccine compositions is described in more detail inthe previous examples. Mice were vaccinated with A/PR/8/34 (PR8; NCBIaccession no. ABO21709; SEQ ID NO:7) whole inactivated virus (WIV). Serawas collected pre-challenge and at 4 weeks post-vaccination. Serasamples were titered in ELISA coated with 2 μg/ml hemagglutinin (HA)from either A/CA/07/09 (H1N1) or A/HK/156/97 (H5N1). FIGS. 32A, 32B, 33Aand 33B show development of broadly reactive α-HA IgG antibodies insera. The amino acid sequence of the HA of A/PR/8/34 (PR8) is NCBIaccession no. ABO21709 (SEQ ID NO:7). The amino acid sequence of the HAof A/CA/07/09 (H1N1; NCBI accession no. AFM72832; SEQ ID NO:8) has 81.3%identity with HA PR8. FIG. 32 shows the inter-strain reactivity betweenPR8 and H1N1. The amino acid sequence of the HA of A/HK/156/97 (H5N1;NCBI accession no. AAC34263; SEQ ID NO:8) has 81.3% identity with HA ofPR8. FIG. 33 shows the inter-strain reactivity between PR8 and H5N1.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference. In the event that anyinconsistency exists between the disclosure of the present applicationand the disclosure(s) of any document incorporated herein by reference,the disclosure of the present application shall govern. The foregoingdetailed description and examples have been given for clarity ofunderstanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed, for variations obvious to one skilled in the art will beincluded within the invention defined by the claims. All headings arefor the convenience of the reader and should not be used to limit themeaning of the text that follows the heading, unless so specified.

1. (canceled)
 2. A vaccine composition comprising a peptide hydrogel ofthe peptide scaffold RADARADARADARADA (SEQ ID NO:2), a hepatitisantigen, and a TLR9 agonist, wherein the vaccine composition is a liquidat room temperature, non-physiological pH, and/or non-physiological saltconcentrations and a gel at physiological pH, physiological saltconcentrations and/or physiological temperatures.
 3. The vaccinecomposition of claim 2, wherein the vaccine composition gels afteradministration to a subject.
 4. The vaccine composition of claim 2,wherein the TLR9 agonist comprises a CpG oligodeoxynucleotide (ODN). 5.The vaccine composition of claim 3, wherein the hepatitis antigencomprises a hepatitis A, hepatitis B, or hepatitis C antigen.
 6. Thevaccine composition of claim 3, wherein the hepatitis antigen comprisesa recombinant Hepatitis B antigen (rHepBag).
 7. The vaccine compositionof claim 3, wherein the vaccine composition is lyopholized.
 8. A methodof producing an immune response in a subject, the method comprisingadministering a vaccine composition of claim 3 to the subject.
 9. Amethod of vaccinating a subject to hepatitis B, the method comprisingadministering a vaccine composition of claim 3 to the subject.
 10. Themethod of claim 8, wherein the subject is human.
 11. The method of claim9, wherein the subject is human.
 12. The method of claim 8, whereinadministration of the composition comprises subcutaneous (sc) delivery,intramuscular (im) delivery, intradermal delivery, transdermal delivery,mucosal delivery, intravaginal delivery, intrarectal delivery,intraperitoneal delivery, inhalation delivery, and/or aerosol delivery.13. The method of claim 9, wherein administration of the compositioncomprises subcutaneous (sc) delivery, intramuscular (im) delivery,intradermal delivery, transdermal delivery, mucosal delivery,intravaginal delivery, intrarectal delivery, intraperitoneal delivery,inhalation delivery, and/or aerosol delivery.